CN104974983A - 用于授精的精子悬浮液 - Google Patents
用于授精的精子悬浮液 Download PDFInfo
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- CN104974983A CN104974983A CN201510400906.7A CN201510400906A CN104974983A CN 104974983 A CN104974983 A CN 104974983A CN 201510400906 A CN201510400906 A CN 201510400906A CN 104974983 A CN104974983 A CN 104974983A
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- sperm
- combination
- antiviral compounds
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/52—Sperm; Prostate; Seminal fluid; Leydig cells of testes
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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Abstract
本文公开了以精子分散体系的形式贮存分选及未分选精子的方法,所述精子分散体系具有的运动性相对于体内射出精子下降。该分散体系中不运动的精子更能耐受贮存、运输和授精过程伴随的困难。本文还公开了提供此类精子分散体系来授精雌性哺乳动物的方法,以及包含该精子分散体系的组合物和组合。
Description
本申请是申请人于2005年3月29日提交的题为“用于授精的精子悬浮液”的中国专利申请200580017376.2的分案申请。
技术领域
本发明主要涉及贮存精子细胞的方法。更具体地,本发明涉及贮存运动性比体内射出精子降低的分选及未分选精子的方法,提供该精子分散体系用于授精雌性哺乳动物的方法,利用该精子分散体系授精雌性哺乳动物的方法。
背景技术
通过人工授精(AI)使动物受精以及体外受精后的胚胎移植已经是成熟的技术。由于用于这些操作的精子的存活力和运动性会影响其结局(即,该受精和授精过程是否能成功产生后代),精子细胞能够耐受授精过程中通常伴随的困难(包括如细胞的收集、贮存和运输)至关重要。
例如,在家畜产业中,常希望可对繁殖结果产生影响,而使其后代具有一种或多种优选性状的能力,如得到特定性别的后代。为达到这样的结局,从雄性哺乳动物收集精液样品,以染料染色,然后分选为带有X和Y染色体的细胞,任选在冰冻或冷却状态下贮存一段时间,然后运输至育种地点,最终在该处对雌性哺乳动物授精。这该过程中的每一步均对于精子细胞产生压力,导致精子细胞的存活力或运动性,特别是前向性运动性(progressive motility)降低。
Salisbury等人描述了将公牛射出的精液直接收集至稀释液中的技术,所述稀释液抑制细胞的运动性并防止其从周围精浆中吸收糖类。当收集射出精液至该稀释液中且将液体上的气相替换为100%的CO2时,射出精液内的细胞会不再运动。只要将细胞留存于该稀释液中且排除空气,该细胞就可在室温下几小时内保持不运动,5℃下至少约8天保持不运动。
发明概述
本发明的多方面之一是在贮存、运输或受精中减少精子运动的方法,从而保护精子免受这些过程中的困难。因此在本发明的一个实施方案中,形成了包含精子和下调精子糖类摄取的组合物的精子分散体系(有时也称为精子悬浮液)。
简言之,本发明因此涉及贮存未分选精子的方法,所述方法包括形成包含精子、诱导精子不运动的组合物以及抗生素的精子分散体系,以及贮存该精子分散体系。
本发明还涉及贮存分选的精子的方法,所述方法包括形成包含精子、诱导精子不运动的组合物的精子分散体系,将该精子分散体系分选为分离群体,并于约-4℃至约30℃的温度下贮存一个群体,其中所述群体之一中的精子包括至少约65%带有X染色体的精子细胞或至少约65%带有Y染色体的精子细胞。
本发明还涉及授精雌性哺乳动物的方法,该方法包括用精子分散体系授精雌性哺乳动物,所述精子分散体系包含不运动的精子和诱导精子不运动的组合物。
本发明还涉及提供新鲜精子分散体系来授精雌性哺乳动物的方法,该方法包括形成包含精子和诱导精子不运动的组合物的精子分散体系、将该精子分散体系置于容器中用以进行远距离运输,并在形成精子分散体系后的约24小时内将该容器内的精子分散体系进行远距离运输。
本方面还涉及包括用于授精雌性哺乳动物的细长容器和精子分散体系的组合,所述精子分散体系包括不运动的精子和诱导精子不运动的组合物。精子分散体系包含在该细长容器中。
本发明还涉及包含不运动的精子、诱导精子不运动的组合物以及冷冻防腐剂的精子分散体系。该精子分散体系中也可包含增强精子存活力的添加剂,例如,抗生素、生长因子、己酸、过氧化氢酶或调节胞内和/或胞外氧化/还原反应的组合物。
本发明的其他方面和特征部分是显而易见的,部分将于此后说明。
发明详述
出人意料的是,已经证明相对于体内射精精子(相同种系),运动性降低的精子,更具体而言是运动性暂时降低的精子,往往不必冷冻就更能耐受贮存和运输过程。大约从来源哺乳动物收集精子时起,就可以将精子保存在运动性降低的状态下,直至用该不运动精子授精雌性哺乳动物。因此,在优选实施方案中,可以制备特定浓度的新鲜未冰冻精子(分选或未分选的)用于贮存、运输或受精,所述精子相对于冰冻后解冻的相同浓度的精子而言,其中活细胞的数目增加或可运动精子的数目增加。
精子分散体系中精子存活力的增加,使得可以在受精或授精过程中可使用包含较低密度精子的精子分散体系等份;这是由于一等份的分散体系包含的活的、前向运动性细胞的百分比(例如人工授精解冻液内包含的细胞百分比)更高,因而授精卵子的潜能也更大。同理,由于受精或授精方法所需的等份分散体系中精子细胞密度更低,精子分散体系形式的单次射精精液可以分为更多等份用于受精或授精方法。
根据本发明的方法,形成包含精子和一种或多种抑制精子运动性的组合物的分散体系;该运动性受抑的状态有时被称为不运动或精子静止。一般而言,该分散体系包含精子的密度为至少约1×103精子/ml分散体系,更优选密度为至少约0.1×106精子/ml分散体系,但一般不超过约5×1010精子/ml分散体系,更优选密度不超过5×108精子/ml分散体系。例如,在一个实施方案中,悬浮液中可包含“相对低”密度的精子,即精子密度少于约1×107精子/ml悬浮液,优选少于约1×106精子/ml悬浮液,更优选约1×103至约5×106精子/ml悬浮液,更优选约1×103至约1×106精子/ml悬浮液,甚至更优选1×104至约1×105精子/ml悬浮液,最优选约1×105精子/ml悬浮液。在另一实施方案中,悬浮液中可包含“中间”密度的精子,即密度为约1×107至约1×108精子/ml悬浮液。在另一实施方案中,悬浮液中可包含“相对高”密度的精子,即精子密度为至少约1×108精子/ml悬浮液,优选约1×108至约5×1010精子/ml悬浮液,更优选约1.5×108至约2×1010精子/ml悬浮液,甚至更优选1.5×108至约2×108精子/ml悬浮液,更加优选约1.5×108精子/ml悬浮液。因此,例如,在一个实施方案中,该分散体系中可包含至少约0.04×106、至少约1×106、至少约1.5x106、至少约2x106、至少约3×106、至少约0.5×107、至少约1×107、至少约2×107、至少约3×107、至少约4×107、至少约5×107、至少约6×107、至少约7×107、至少约8×107、至少约9×107、或甚至至少约12×107精子/ml分散体系。在另一实施方案中,悬浮液中可包含小于约9×105、小于约7×105、小于约5×105、小于约2×105、小于约1×105、小于约1×104、或甚至小于约1×103精子/ml悬浮液。
精子的密度可取决于若干因素,包括如获取精子的哺乳动物种系。例如,牛精子分散体系密度可较高,但一般量较小,例如0.5×106精子/ml至约8×107精子/ml,量约0.5ml至约25ml。然而猪精子分散体系可能密度较低但一般量较大,例如0.04×106精子/ml至约1×107精子/ml,量为约50ml至约250ml。
精子分散体系中的精子密度可因个体哺乳动物或种系特异性因素而有所差异。此类因素的实例包括如哺乳动物不同种系间的差异、某一种系哺乳动物间的差异,甚至同一动物不同次射精间的差异。
也可人工操作精子分散体系中精子的密度,以获得具有特定精子密度的分散体系。对于精子分散体系中的精子密度的操作(如在授精解冻物中进行),可取决于若干因素,例如分散体系的贮存温度、贮存期的长度、精子分散体系中的精子是分选过的还是未分选过的、收集精子的雄性哺乳动物的种系、收集精子的哺乳动物的生育力,以及将接受授精的雌性哺乳动物的种系。
随后富集或分选精子细胞的方法也可影响精子分散体系中的精子密度。例如,可使用如下文详述的流式细胞术分选精子细胞。在这种情况下,缓冲精子悬浮液一般含“中间”或“相对高”密度的精子。其他分选或富集技术更利于在精子密度更低时使用,例如标有标记(如此处所述的染料和标记)的“相对低”密度的精子。
简单浓缩(例如通过离心)精子也会影响精子分散体系中的精子密度。在该情况下,可将分散体系大致分为常说的沉淀(包含最少量液体的细胞团)和上清液(可溶性的液体级分)。随后可以在不干扰沉淀的情况下倾析上清液,从而得到相对密实的包含最少量抑制性缓冲液的精子细胞沉淀,其作用是减少分散体系的体积而不改变分散体系的成分。这样,沉淀中的精子细胞保持不运动的状态。
在优选实施方案中,本发明分散体系中的精子在某些方面表现出附睾精子的特征性行为;例如,精子不运动,相对于刚射出精子而言其内呼吸速率更低而有氧糖酵解的速率更高。有利的是,受抑精子具有与一种或多种抑制剂分离后,运动性能够表现出射出精子的特征性行为(而不具有附睾精子的特征性行为);而在一个实施方案中,其运动性和呼吸均能够表现出射出精子的特征性行为。
例如在一个实施方案中,通过HTM-IVOS精子分析(Hamilton-ThorneHTM-IVOS计算机辅助精子分析系统,Hamilton-Thorne Research,Beverly MA)测量,运动抑制剂可使分散体系中精子细胞的路径速度、前向性速度或此两者相对于相同种系刚射出精液中精子细胞的路径速度、前向性速度或此两者,降低至少约50%。优选通过HTM-IVOS精子分析测量,运动抑制剂可使分散体系中精子细胞的路径速度、前向性速度或此两者相对于相同种系刚射出精液中精子细胞的路径速度、前向性速度或此两者,降低至少约60%。更优选通过HTM-IVOS精子分析测量,运动抑制剂可使分散体系中精子细胞的路径速度、前向性速度或此两者相对于相同种系刚射出精液中精子细胞的路径速度、前向性速度或此两者,降低至少约70%。进一步优选通过HTM-IVOS精子分析测量,运动抑制剂可使分散体系中精子细胞的路径速度、前向性速度或此两者相对于相同种系刚射出精液中精子细胞的路径速度、前向性速度或此两者,降低至少约80%。甚至更优选通过HTM-IVOS精子分析测量,运动抑制剂可使分散体系中精子细胞的路径速度、前向性速度或此两者相对于相同种系刚射出精液中精子细胞的路径速度、前向性速度或此两者,降低至少约90%。甚至更优选通过HTM-IVOS精子分析测量,运动抑制剂可使分散体系中精子细胞的路径速度、前向性速度或此两者相对于相同种系刚射出精液中精子细胞的路径速度、前向性速度或此两者,降低至少约95%。最优选通过HTM-IVOS精子分析测量,运动抑制剂可使分散体系中精子细胞的路径速度、前向性速度或此两者相对于相同种系刚射出精液中精子细胞的路径速度、前向性速度或此两者,降低至少约99%。
除抑制性缓冲液外或可替代其的是,可单独降低精子细胞的温度或紧邻精子细胞的环境温度(即精子分散体系)以影响细胞的运动性。此种温度降低一般会增加不运动性。此外,例如,精子细胞或精子分散体系温度的降低会使用于诱导不运动性的抑制剂浓度降低。因此,精子分散体系可处于不超过5℃的温度下;优选在约0℃至约5℃之间;更优选为约3℃至约5℃之间;而最优选为5℃。或者精子分散体系的温度可介于约4℃至约50℃范围内;优选为约7℃至约43℃;更优选为约10℃至约39℃;进一步优选为约15℃至约30℃;甚至更优选为约17℃至约25℃;而最优选为约18℃。然而,优选不将精子细胞暴露于会对细胞存活力造成实质性不良影响的温度下。
抑制剂可以为一系列对精子运动性具有抑制作用的组合物中的任意一种。此类组合物包括如钠/钾ATP酶抑制剂,如乌本苷;包含钾离子的组合物;以及包含钾离子和钠离子的组合物。例如,分散体系中相对高浓度的钾离子会抑制精子运动性。因此一般而言,优选分散体系包含钾离子源且分散体系中的钾浓度为至少约0.05摩尔/升。更优选钾离子浓度为至少约0.05摩尔/升至约0.5摩尔/升。更优选钾离子浓度为至少约0.1摩尔/升至约0.3摩尔/升。最优选钾离子浓度为约0.173摩尔/升。此类悬浮液一般(但非必须)也包含钠离子源。当包含钠离子时,钾和钠各自的摩尔比一般等于或大于1:1,但摩尔比一般不超过8:1。优选钾和钠的摩尔比为至少约1.25:1。更优选钾和钠的摩尔比为至少约1.5:1。进一步优选钾和钠的摩尔比为至少约1.75:1。更优选钾和钠的摩尔比为至少约1.78:1。在一个具体实施方案中,钾和钠的摩尔比为至少约2:1。而在另一实施方案中,钾和钠的摩尔比为至少约3:1。在另一实施方案中,钾和钠的摩尔比为至少约4:1。在另一实施方案中,钾和钠的摩尔比为至少约5:1。在另一实施方案中,钾和钠的摩尔比为至少约6:1。在另一实施方案中,钾和钠的摩尔比为至少约7:1。在另一实施方案中,钾和钠的摩尔比为至少约8:1。
精子分散体系中还可额外包含能够提高或有助于抑制精子活动性的离子或二氧化碳源。该二氧化碳源可为分散体系的组成成分的形式,例如一种或多种碳酸盐;其也可为分散体系上方二氧化碳正分压大于周围空气中天然二氧化碳分压的气氛的形式。或者,二氧化碳源的形式可为分散体系的组成成分,以及分散体系上方二氧化碳正分压大于周围空气中天然二氧化碳分压的气氛的组合。
在目前优选的一个实施方案中,精子分散体系包含NaHCO3和KHCO3,因此提供了钾离子和钠离子源以及二氧化碳分压。例如,在目前优选的一个实施方案中,分散体系包含NaHCO3和KHCO3水性溶液,优选NaHCO3、KHCO3和C6H8O7·H2O的水溶液;一般而言,分散体系中KHCO3的浓度可为至少约0.05摩尔/升。更优选KHCO3的浓度为至少约0.05摩尔/升至约0.5摩尔/升。进一步优选KHCO3的浓度为至少约0.1摩尔/升至约0.3摩尔/升,最优选KHCO3的浓度为至少约0.173摩尔/升。当包含NaHCO3时,KHCO3与NaHCO3的摩尔比可为上文所述的有关钾和钠的摩尔比。
当分散体系包含C6H8O7·H2O时,KHCO3与NaHCO3间的摩尔比可如上所述。KHCO3与C6H8O7·H2O间的摩尔比一般等于或大于1:1,但一般不会超过摩尔比8:1。优选KHCO 3与C6H8O7·H2O间的摩尔比为至少约1.25:1。更优选KHCO3与C6H8O7·H2O间的摩尔比为至少约1.5:1。进一步优选KHCO3与C6H8O7·H2O间的摩尔比为至少约1.75:1。在一个具体实施方案中,KHCO3与C6H8O7·H2O间的摩尔比为至少约1.78:1.在另一具体实施方案中,KHCO3与C6H8O7·H2O间的摩尔比为至少约2:1。而在另一实施方案中,KHCO3与C6H8O7·H2O间的摩尔比为至少约3:1。在另一实施方案中,KHCO3与C6H8O7·H2O间的摩尔比为至少约4:1。在另一实施方案中,KHCO3与C6H8O7·H2O间的摩尔比为至少约5:1。在另一实施方案中,KHCO3与C6H8O7·H2O间的摩尔比为至少约6:1。在另一实施方案中,KHCO3与C6H8O7·H2O间的摩尔比为至少约7:1。在另一实施方案中,KHCO3与C6H8O7·H2O间的摩尔比为至少约8:1。在一个特别优选的实施方案中,利用如Salisbury&Graves,J.Reprod.Fertil.,6:351-359(1963)中所公开,含0.097摩尔/升NaHCO3、0.173摩尔/升KHCO3、0.090摩尔/升C6H8O7·H2O的水溶液的抑制性缓冲液形成悬浮液。只要精子细胞暴露于一种或多种运动抑制剂,它们一般都会保持静止。
迄今为止的实验证据还提示,如果将精子细胞分散体系保存在降低或防止氧气向该分散体系扩散的气氛中,可以改善精子细胞的整体健康和其他生命特征。通过用如二氧化碳、氮气或其他惰性气体分压比周围大气增高的气氛来代替精子分散体系上方的气氛,可以达到这一目的。在优选实施方案中,分散体系上方的气氛中二氧化碳分压为至少约0.000latm,但一般小于约5atm大气压。在一个实施方案中,二氧化碳分压为约0.5atm至约2atm大气压;在另一实施方案中,二氧化碳分压为约0.9atm至约2atm大气压;在另一实施方案中,二氧化碳分压为约0.95atm至约2atm大气压。
可以将细胞与运动性抑制剂分离并将其暴露于空气中,可使静止或不运动细胞还原为活性状态。此外,还可以通过在生理盐水中(Salisbury等人,1963)或如TCA缓冲液或PBS的缓冲液中稀释细胞来诱导活性状态的起始。一般而言,根据HTM-IVOS精子分析测量,至少约20%、优选至少约50%、更优选至少约60%、进一步优选至少约70%、甚至更优选至少约80%、甚至更优选至少约90%、更优选至少约95%,而最优选至少约99%回复活性状态的细胞(即再活化细胞)将具有路径速度、前向性速度或此两者;所述速度至少约分别为与运动性抑制剂结合前的精子细胞(即刚射出的精子细胞)的路径速度、前向性速度或此两者的至少约50%,优选至少约60%,更优选至少约70%,进一步优选至少约80%,甚至更优选至少约90%,甚至更优选至少约95%,而最优选至少约99%。
本方法可用于贮存、运输以及用分选及未分选精子授精。若根据本发明的方法贮存未分选精子,可将精子直接收集至包含运动性抑制剂的容器中以形成精子分散体系。该精子分散体系中也可包含至少约一种抗生素。随后可将该精子分散体系贮存所需的时间。
若根据本发明的方法贮存分选的精子,可将精子直接收集至包含运动性抑制剂的容器中以形成精子分散体系。一旦完成收集,可按照多步骤的方法分选分散体系中的精子。一般而言,细胞分选方法包括一系列分离步骤,即收集细胞的染色、分选细胞、收集分选的细胞,以及任选冷冻伸展(cryoextension)分选的细胞。随后可将该分选细胞贮存所需的时间。有利的是,运动性抑制剂可包含在精子分散体系中,或用于细胞分选方法步骤中的一步或多步中。
I.细胞样品的收集
不管本发明的方法是用于贮存分选还是未分选的精子,可以收集牛、猪(porcine)、马、猪(swine)或其它哺乳动物完整的活的精子细胞并将其与运动性抑制剂接触。已知多种收集有活精子的方法,其包括如手握法、使用人工阴道以及电刺激射精。例如,可以将一般每毫升包含5亿至100亿精子细胞的牛精子样品,直接从来源哺乳动物或相同种系的多个来源哺乳动物体内收集于包含运动性抑制剂的容器中,以形成精子分散体系。也可以将精子样品收集于空容器中,随后在收集的几分钟至几小时内将其与运动性抑制剂接触以形成精子悬浮液。
除缓冲物外,精子分散体系中也可包含一系列添加剂以增强精子存活力。示例性添加剂包括固醇、脂质和脂肪酸、蛋白质源、抗生素、生长因子、己酸、过氧化氢酶、Caprogen(己酸、过氧化氢酶和5%卵黄),以及调节胞内和/或胞外氧化/还原反应的组合物。
示例性蛋白质源包括卵黄、卵黄提取物、乳(包括热匀浆和脱脂乳)、乳提取物、大豆蛋白、大豆蛋白提取物、血清白蛋白、牛血清白蛋白、人血清替代增补物、精液蛋白(如全精浆或精浆提取物,见如Parks等人.,Sperm Membrane Phospholipid Peroxidation and Fragmentation:Effects onSperm Function and Role of Seminal Plasma PAF-Acetylhydrolase,Proceedings of the 16th Technical Conference on Artificial Insemination&reproduction,1996,其内容在此处引用作为参考),及其组合。白蛋白和特别是牛血清白蛋白(BSA),是优选的蛋白质源。例如若精子分散体系中包含BSA,其含量可少于约5.0%(w/v),优选少于约2%(w/v),更优选少于约1%(w/v),而最优选为约0.1%(w/v)的量。
单独使用蛋白质源(如BSA),可起始分散体系中百分之一的精子细胞的获能过程。该过程优选发生于雌性生殖道内。因此,为了抑制稀释期间以及随后的染色和分选中获能的起始,精子分散体系中可包含备选的蛋白质源或蛋白质替代物。所述的备选蛋白质源或蛋白质替代物除了具有能够抑制精子悬浮液中更大百分比的细胞起始获能,还具有典型蛋白质源(如BSA)的有益作用。备选蛋白质源的实例包括人血清替代增补物(SSS)(Irvine Scientific,Santa Ana,CA)以及胆固醇强化的BSA,而蛋白质替代物的实例包括聚乙烯醇,例如一般分子量为约30,000至约60,000的低至中粘度聚乙烯醇。一般而言,若包含这些组合物,其含量与上述BSA的量相同;缓冲物或缓冲溶液中总白蛋白含量一般不超过约5.0%(w/v)。
调节胞内和/或胞外氧化/还原反应的示例性组合物包括如丙酮酸盐、维生素K、硫辛酸、谷胱甘肽、黄素、醌类、超氧化物歧化酶(SOD)以及SOD模拟物。若精子分散体系包含此类组合物,则其浓度足以产生保护性作用而不会对精子健康产生不良影响。示例性浓度范围包括从约10μM至约20mM,取决于诸如所用的具体组合物或分散体系中精子的浓度等因素。例如,精子分散体系中丙酮酸盐的浓度可为约1mM至约20mM,优选为约5mM至约15mM,更优选为约10mM。精子分散体系中维生素K的浓度可为约1μM至约100μM,优选为约10μM至约100μM,更优选为约100μM。精子分散体系中硫辛酸的浓度可为约0.1mM至约1mM,优选为约0.5mM至约1mM,而更优选为约1mM。
精子分散体系中可包含抗生素以抑制细菌的生长。示例性抗生素包括如,泰乐菌素、庆大霉素、林可霉素、壮观霉素、利高霉素(林可霉素羟氯化物-壮观霉素)、青霉素、链霉素、替卡西林(ticarcillin)、多粘菌素B或其任意组合物。若包含抗生素,则其浓度可为每毫升精液中约50μg至约800μg(不论精液是否是纯净的、缓冲性的或包含其他物质,例如此处提及的任一种添加剂)℃ertified Semen Services(CSS)和美国国家动物育种协会(NAAB)已经颁布了有关抗生素在精子收集和使用中应用的纲要。
可以向精子分散体系中加入生长因子,以协助维持精子细胞的存活力。示例性生长因子包括如转化生长因子("TGF)(例如TGFβ-1和TGFβ-2),以及胰岛素样生长因子("IGF")(例如IGF-1)。一般而言,精子分散体系中的TGF可以TGFβ-1的形式出现,其浓度为约0.l ng/L至约10μg/L,或为TGFβ-2的形式,其浓度为约0.1ng/L至约200ng/L;而IGF在精子分散体系中可为IGF-1形式,其浓度为约0.1ng/L至约50μg/L。此类生长因子的用途为本领域所公知,并公开于如美国专利申请公开号2003/0157473中,其内容在此处引用作为参考。
一旦收集了细胞,可将以静止状态保存所需时间,或者在数小时内使用。两者中任一情况下,细胞都可用于如染色过程、分选过程或育种过程。
A.收集细胞的分选
(i)细胞染色
运动性抑制剂可用于使细胞在细胞染色期间不运动。一般而言,精子细胞染色方法中可包括形成染色混合物(有时称为标记混合物),其中包含完整的活精子细胞、运动性抑制剂和染料(有时称为标记)。在本发明的这一方面,可以将运动性抑制剂与精子细胞接触以形成精子分散体系,然后将精子分散体系与DNA选择性染料相接触。在该实施方案中,精子来源可为纯精液,或者为通过离心或使用其他手段分离精液级分而得到的包含精子的精液衍生物。
在备选实施方案中,染料可与运动性抑制剂相组合,从而形成染料溶液。因此如纯固体(包括可自由流动的粉末)或液体组合物形式的染料可与抑制剂组合形成染料溶液,所述溶液可随后与纯精子、精子分散体系或含精子的精液衍生物组合。
任何情况下,只要精子细胞保存在抑制剂中,一般都会保持静止(Salisbury等人,1963)。然而,优选将染色混合物保存在二氧化碳分压相对于周围空气而言增高的大气中;例如,一般优选染色混合物上方的大气中含99%+的CO2。
可将染色混合物的pH保持在一定pH值范围内的任意一点;一般而言,该范围为约5.0至约9.0。例如,可使染色混合物保持在“微酸性”pH下,即约5.0至约7.0。在该实施方案中,优选pH为约6.0至约7.0,更优选为约6.0至约6.5,而最优选为约6.2。备选地,可使染色混合物保持在“微碱性”pH下,即约7.0至约9.0。在该实施方案中,优选pH为约7.0至约8.0,更优选为约7.0至约7.5,而最优选为约7.35。
如以前在美国专利号5,135,759和WO 02/41906中所述,可使用一种或多种可经UV或可见光激发的DNA选择性染料可以形成染色混合物,其内容均在此处引用作为参考。示例性紫外光(UV)激发的选择性染料包括Hoechst33342和Hoechst33258,二者均可从Sigma-Aldrich(St.Louis,MO)购得。示例性可见光激发的染料包括购自Molecular Probe,Inc.(Eugene,OR)的SYBR-14,以及WO02/41906中所描述的bisbenzimide-缀合物6-{[3-((2Z)-2-{[1-(二氟硼烷基)-3,5-二甲基-lH-吡咯-2-基]亚甲基}-2H-吡咯-5-基)丙酰基]氨基}-N-[3-(甲基{3-[({4-[6-(4-甲基哌嗪-l-基)-lH,3'H-2,5'-二苯并咪唑-2'-基]苯氧基}乙基)氨基]丙基}氨基)丙基]己酰胺(“BBC”)。这些染料中的每一种都可以单独使用或者联合使用;或可以将其他具有细胞渗透性的UV和可见光激发的染料单独或与前述染料联合使用,只要所述染料在可进行别处所述分选或富集的浓度下不会对精子细胞的存活力产生不可接受的不良影响。
还可利用荧光聚酰胺形成染色混合物,更具体而言是利用缀合有荧光标记或报告子的聚酰胺。此类标记在结合于核酸时会发出荧光。连接有荧光标记或报告子的聚酰胺实例包括,如Best等人,Proc.Natal.Acad.Sci.USA,100(21):12063-12068(2003);Gygi等人,Nucleic Acids Res.,30(13):2790-2799(2002);美国专利号5,998,140;美国专利号6,143,901以及美国专利号6,090,947中所公开的那些,每一篇的内容均在此处引用作为参考。
荧光核苷酸序列也可用于标记精子细胞。此类核苷酸序列在与包含靶序列或互补序列的核酸杂交时会发出荧光,但在非杂交状态时则不会发出荧光。此类核苷酸公开见于如美国专利申请公开号2003/0113765(此处引用作为参考)。
性别特异性抗体也可用于标记染色混合物中的精子细胞。例如在该实施方案中,性别特异性抗体可缀合于荧光部分(或等价报告分子)。由于抗体结合的抗原只存在于带有X染色体或Y染色体的细胞上,根据其荧光(相对于无荧光的未标记细胞)能够选择性地鉴定此类细胞。此外,可以同时使用分别与不同荧光部分相连的一种以上性别特异性抗体。从而能根据带有X染色体或Y染色体的细胞间的不同荧光,对之进行区分。
也可使用发光的色彩选择性纳米晶体标记染色混合物中的精子细胞。这些颗粒也称为量子点,正如美国专利号6,322,901和美国专利号6,576,291所述为本领域所公知(均在此处引用作为参考)。这些纳米晶体已被缀合于多种生物材料,包括如肽、抗体、核酸、链霉亲和素及多糖(见如美国专利号6,207,392、6,423,551、5,990,479和6,326,144,均在此处引用作为参考),且已用于检测生物靶标(见如美国专利号6,207,392和6,247,323,两者均在此处引用作为参考)。
染色混合物中DNA选择性染料或任何其他类型染料的浓度优选为一系列变量的函数,所述变量包括细胞对于所选择染料的渗透性、染色混合物的温度、完成染色所需要的时间、精子浓度以及在随后的分选步骤中要求的富集程度。一般而言,优选的染料浓度足以在适度短的时间内达到所需的染色程度,而不会对精子存活力产生实质性不良影响。例如,Hoechst33342、Hoechst 33258、SYBR-14或BBC在混合物中的浓度一般在约0.1μM至约1.0M之间,优选为约0.1μM至约1000μM,更优选为约100μM至约500μM,更优选为约200μM至约500μM,更优选为约300μM至约450μM。因此,在一种染色条件设置下,Hoechst33342的浓度优选为约350μM。在另一种染色条件设置下,Hoechst33342的浓度为约400μM。在另一种染色条件设置下,浓度优选为约450μM。
又例如,荧光聚酰胺的浓度(例如美国申请公开号2001/0002314中所述的那些),一般为约0.1μM至约1mM,优选约1μM至约1mM,更优选约5μM至约100μM,甚至更优选约10μM。
染色混合物中也可任选地包含增强精子存活力的添加剂。示例性添加剂包括如上文“细胞样品的收集”中已经讨论过的抗生素、生长因子或调节胞内和/或胞外氧化/还原反应的组合物。据此向收集液中添加此类添加剂。
染色混合物一旦形成,可以在一系列温度范围中的任意温度下保存;一般而言,温度范围为约4℃至约50℃。例如,可以将染色混合物保存于“相对低”的温度下,即温度为约4℃至约30℃;在该实施方案中,温度优选为约20℃至约30℃,更优选为约25℃至约30℃,最优选为约28℃。或者也可将染色混合物保存于“中间”温度范围内,即温度为约30℃至约39℃;在该实施方案中,温度优选为约34℃至约39℃,更优选为约37℃。此外还可将染色混合物保存于“相对高”的温度范围内,即温度为约40℃至约50℃;在该实施方案中,温度优选为约41℃至约49℃,更优选为约41℃至约45℃,更优选为约41℃至约43℃,最优选为约41℃。对于优选温度的选择一般取决于一系列变量,包括如细胞对所用染料的渗透性、染色混合物中染料的浓度、细胞在染色混合物中留存的时间、以及在分选或富集步骤中所需的富集程度。
精子细胞摄取染色混合物中的染料可以持续一段足够长的时间,以达到所需程度的DNA染色。所述时间一般足以使染料结合于精子细胞的DNA,从而能根据带有X及Y染色体的精子细胞间不同的且可测量的荧光强度,将两者分选或富集出来。一般而言,这一时间不会超过约24小时;优选不超过约10小时,更优选不超过2小时,进一步优选不超过约90分钟,甚至更优选不超过约60分钟,而最优选为约5分钟至约60分钟。在具体实施方案中,这一时间为约30分钟。
染色阶段的长度与染色时温度的关系为,染色时间越长,染色温度就可越低。例如,在一个实施方案中,染色可发生于“相对低”的温度下,持续约3小时至约24小时。或者,染色可发生于“中间”的温度下,持续约半小时至约3小时。此外,染色可发生于“相对高”的温度下,持续约10分钟至约90分钟。在具体的实施方案中,染色可发生于约4℃下持续约24小时。在另一实施方案中,染色可发生于约18℃下持续约4小时。在另一实施方案中,染色可发生于约41℃下持续约30分钟。
因此,在一个实施方案中,形成包含精子细胞、运动性抑制剂和浓度为约100μM至约450μM的染料的染色混合物,且将该染色混合物在约41℃的温度下保持一段时间。在另一实施方案中,运动性抑制剂于每25ml纯水包含0.204g NaHCO3、0.433g KHCO3和0.473g C6H8O7·H2O(NaHCO30.097摩尔/升、KHCO3 0.173摩尔/升、C6H8O7·H2O 0.090摩尔/升的水溶液)。
在另一实施方案中,形成包含精子细胞、运动性抑制剂和浓度为约100μM至约450μM的染料的染色混合物,且将该染色混合物在约28℃的温度下保持一段时间。在另一实施方案中,该时间长度为约3小时。在另一实施方案中,运动性抑制剂为每25ml纯水包含0.204g NaHCO3、0.433gKHCO3和0.473g C6H8O7·H2O(NaHCO3 0.097摩尔/升、KHCO3 0.173摩尔/升、C6H8O7·H2O 0.090摩尔/升的水溶液)。
(ii)染色细胞的分选或富集
运动性抑制剂也可用于在精子细胞分选过程中使细胞不运动。一般而言,一旦根据本发明将精子染色,可以根据任何已知的可依据荧光分离的方法分选之。常用且公知的方法包括流式细胞术体系(如美国专利号5,135,759、5,985,216、6,071,689、6,149,867及6,263,745;国际专利公开号WO99/33956和WO01/37655及美国专利申请系列号10/812,351(相应的国际专利公开号WO 2004/088283)中举例及描述的那些,其每一篇的内容均在此处引用作为参考)。当根据这些方法分选时,将精子作为样品液引入流式细胞仪的管口中。因此在一个实施方案中,样品液可包包含染色精子细胞和运动性抑制剂。
与之类似,用于在穿行细胞仪时包裹样品液流的鞘液中也可包含运动性抑制剂。一般而言,利用加压空气或注射泵将鞘液引入流式细胞仪的管口。优选的加压空气为二氧化碳或氮气,更优选二氧化碳。备选地,加压空气也可为氮气。
样品液或鞘液中也可任选包含添加剂,如上文“细胞样品的收集”中讨论过的抗生素、调节胞内和/或胞外氧化/还原反应的组合物或生长因子。据此,将这些添加剂中的每一种加入任一种液体中。
备选地,可利用激光控制进行精子分散体系中细胞的分选或富集。这也常称为光学捕获或全息光学捕获。一般而言,高度聚焦的激光(例如通过显微透镜聚焦的光)具有大的强度梯度。光学捕获根据光束的介电常数,利用一束光的梯度力来捕获粒子。为使其能量最低,介电常数大于周围介质的粒子会移动到电场最高的光阱区域。此类装置和方法描述于如WO2004/012133、美国专利号6,416,190及相关申请和专利,其每一篇内容均在此处引用作为参考。根据这些方法分选或富集出的精子细胞的不动性使其易于在流动中排列,也便于激光控制细胞,从而能更有效和准确地分选或富集所需细胞。
因此,可将精子分散体系中的细胞分选为分离的群体,其中所述群体的精子包含一定百分比带有X染色体或带有Y染色体的精子细胞。例如,某一群体的精子可包含至少约65%带有X染色体或Y染色体的精子细胞,至少约70%带有X染色体或Y染色体的精子细胞,至少约75%带有X染色体或Y染色体的精子细胞,至少约80%带有X染色体或Y染色体的精子细胞,至少约85%带有X染色体或Y染色体的精子细胞,至少约90%带有X染色体或Y染色体的精子细胞,或甚至至少约95%带有X染色体或Y染色体的精子细胞。
(iii)分选细胞的收集
一旦完成分选,将分选细胞收集于包含收集液的容器中。一般而言,使用收集液的目的包括提供细胞的液体支持。
在一个实施方案中,收集液中可包含运动性抑制剂。收集液中也可任选包含固醇、脂质、脂肪酸或蛋白质源。若含纳于收集液内,则固醇、脂质和脂肪酸可为如胆固醇。
若收集液包含蛋白质源,则其可为任何不会干扰精子细胞存活力并与所用的运动性抑制剂相容的蛋白质源。常用蛋白质源的实例包括乳(包括热匀浆和脱脂乳)、乳提取物、卵黄、卵黄提取物、大豆蛋白和大豆蛋白提取物。此类蛋白质的使用浓度可为约1%(v/v)至约30%(v/v),优选约10%(v/v)至约20%(v/v),更优选约10%(v/v)。
任选地,收集液中也可含添加剂,如上文“细胞样品的收集”中讨论过的抗生素、生长因子或调节胞内和/或胞外氧化/还原反应的组合物。据此,将这些添加剂中的每一种加入收集液中。
因此,在某一实施方案中,收集液为包含0.097摩尔/升NaHCO3、0.173摩尔/升KHCO3、0.090摩尔/升C6H8O7·H2O以及10%(v/v)卵黄的水溶液,pH为约6.2,更优选pH为约7.0,甚至更优选为约6.5。优选将收集液保存在富集的二氧化碳分压高于空气的气氛中;例如,该气氛中二氧化碳分压可大于0.9,进一步优选0.95,更优选0.99。
可将分选细胞收集至包含或包被有冷冻伸展剂的容器中,以代替更常规的收集液的使用。因此,在一个具体实施方案中,分选细胞收集于包含运动性抑制剂的冷冻伸展剂中。在另一实施方案中,分选的细胞收集于包含运动性抑制剂、水、(Minitube,Verona,WI,每升包含60ml甘油、24.2g tris、13.8g柠檬酸、10.0g果糖、泰乐菌素5mg/100ml、庆大霉素25mg/100ml、壮观霉素30mg/100ml及林可霉素15mg/100ml)、卵黄,以及任选丙酮酸盐。在另一实施方案中,收集液为包含0.097摩尔/升NaHCO3、0.173摩尔/升KHCO3、0.090摩尔/升C6H8O7·H2O水溶液的冷冻伸展剂,且每75ml水中含25g25g卵黄和10mM丙酮酸盐。在另一实施方案中,收集液为包含0.097摩尔/升NaHCO3、0.173摩尔/升KHCO3、0.090摩尔/升C6H8O7·H2O水溶液的冷冻伸展剂,且每75ml水中含25g25g卵黄。
(iv)分选细胞的冷冻伸展
一旦完成精子的分选并收集其至收集容器内,这些精子即可用于对雌性哺乳动物授精。这几乎不需要对精子进行其他处理就可以立即进行。在这种情况下,可以将精子以其现状贮存于一段必要时间,以例如将精子运输到进行授精的地点。因此,可于如收集液中贮存并运输精子。同理,可将精子于运动性抑制剂中浓缩至适合某具体哺乳动物种系的密度,例如密度为约50×106精子/ml至约120×106精子/ml,随后贮存并运输。选择的浓度取决于如下文“受精”中所讨论的那些,其包括获取细胞的哺乳动物种系。这种基于获得精子的哺乳动物种系的一系列密度为本领域技术人员所熟知。如下文详细所述,在任何情况下,贮存或运输期间的精子都保持不运动。
同样,也可以将精子冷却或冷冻以备后用。在这种情况下,加入冷冻伸展剂以最大程度地降低冷却或冷冻对存活力或解冻后运动性的影响,对精子有益。
运动性抑制剂可用于使冷冻伸展剂中的细胞不运动。一般而言,冷冻伸展剂包含运动性抑制剂、蛋白质源和冷冻保护剂。若包含,也可加入蛋白质源以提供细胞支持。蛋白质源可为不会干扰精子细胞存活力并与运动性抑制剂相容的任何蛋白质源。常用蛋白质源的实例包括乳(包括热匀浆和脱脂乳)、乳提取物、卵黄、卵黄提取物、大豆蛋白和大豆蛋白提取物。此类蛋白质的使用浓度可为约10%(v/v)至约30%(v/v),优选约10%(v/v)至约20%(v/v),更优选约20%(v/v)。
冷冻伸展剂中优选包含冷冻保护剂,以减轻或避免冷冻休克或以保持精子的生育力。本领域已知众多冷冻保护剂。适于与给定伸展剂共同使用的冷冻保护剂可有多种选择,取决于待冷冻的精子来源于哪一种系。合适的冷冻保护剂的实例包括如,甘油、二甲基亚砜、乙二醇、丙二醇、海藻糖、及其组合。若包含冷冻保护剂,则其在冷冻伸展剂中的量一般为约1%(v/v)至约15%(v/v),优选其量为约5%(v/v)至约10%(v/v),更优选其量为约7%(v/v),最优选量为约6%(v/v)。
在具体实施方案中,冷冻伸展剂包含运动性抑制剂、水、卵黄和任选丙酮酸盐。而在另一实施方案中,冷冻伸展剂为含0.097摩尔/升NaHCO3、0.173摩尔/升KHCO3、0.090摩尔/升C6H8O7·H2O的水溶液,且每75ml水中含25g25g卵黄和10mM丙酮酸盐。
在另一个具体实施方案中,冷冻伸展剂包含运动性抑制剂、水、和卵黄。在另一实施方案中,冷冻伸展剂为含0.097摩尔/升NaHCO3、0.173摩尔/升KHCO3、0.090摩尔/升C6H8O7·H2O的水溶液,且每75ml水中含25g及25g卵黄。
任选地,冷冻伸展剂中也可包含如上文“细胞样品的收集”中讨论过的抗生素、生长因子或调节胞内和/或胞外氧化/还原反应的组合物。据此,将这些添加剂中的每一种加入收集液中。
II.收集细胞的贮存
A.贮存时间
一旦从来源哺乳动物中收集细胞(不论其此后是否任选进行分选),细胞都将贮存一段时间。贮存时间取决于若干因素,包括如贮存细胞时的温度、贮存容器内的细胞数目、细胞为分选的还是未分选的、细胞将用于授精的方法以及受精的雌性哺乳动物。例如,一般而言,贮存细胞数小时,如2、4、8、12或24小时;数日,如1、2、3、4、5、6或7日;数周,1、2、3或4周;或数月,如1、2或3个月。一般而言,可于约5℃至约30℃温度下贮存细胞数小时至数天;可于约-4℃至约5℃温度下贮存细胞数日至数周;可于约-190℃(于液氮蒸汽中)至约-4℃温度下贮存细胞数周至数月。
B.贮存温度
细胞可贮存于一系列不同的温度下。贮存温度的选择取决于若干因素,例如细胞将贮存的时间的长度、贮存容器内的细胞浓度、细胞为分选还是未分选的、细胞将用于授精的方法以及受精的雌性哺乳动物。所有这些因素都会影响在贮存期内仍保持存活力的精子数目。例如,一般而言,细胞可能贮存的时间越长,其可能贮存的温度越低。温度的降低可使更大百分比的贮存细胞在长时期内保持存活。
因此,可将细胞贮存于约-196℃至约30℃的温度下。例如,细胞可贮存于“相对低贮存温度”下,即温度范围约-196℃至约-4℃;在该实施方案中,温度优选为约-12℃至约-4℃,更优选为-10℃至约-4℃,最优选为约-4℃。或者也可将细胞贮存于“中间贮存温度”下,即温度范围约-4℃至约5℃;在该实施方案中,温度优选为约-3℃至约5℃,更优选为约0℃至约5℃,而最优选为5℃。此外,细胞可贮存于“稍高贮存温度”下,即温度范围约5℃至约30℃;在该实施方案中,温度优选为约10℃至约25℃,更优选为约12℃至约23℃,更优选为约15℃至约20℃,而最优选为18℃。有利的是,已经确定将细胞贮存于约18℃的抑制性缓冲物中可以提供额外的益处,即存活和运动精子细胞的数目增多;因为在较高温度或冰点温度(即温度低于0℃)时贮存的困难都可对精子存活力造成有害影响。
C.贮存容器
可将精子分散体系贮存于一系列不同的容器中。由于容器的大小不同,一般而言合适的容器需能容纳精子分散体系;也就是说,制成容器的材料一般不易因接触液体,更具体地为精子分散体系而发生渗漏或变质,不管这一接触发生于容器的内部还是外部。合适容器的实例包括如,烧瓶、烧杯、试管、安瓿,以及其他此类一般由玻璃、塑料或其他类似材料制成的容器。在具体实施方案中,容器为一类用于授精雌性哺乳动物的结构物,例如细长容器。此类细长容器的长度与直径之比一般为约1000:1至约100:1,优选长度与直径之比为约900:1至约200:1,更优选长度与直径之比为约800:1至约300:1,更优选长度与直径之比为约700:1至约400:1,甚至更优选长度与直径之比为约600:1至约400:1,更优选长度与直径之比为约500:1至约400:1,而在具体实施方案中,长度与直径之比为约450:1。此类细长容器的容量一般为约0.1cc至约100cc,使用容器容量的选择取决于获得精子的哺乳动物种系。例如,此类细长容器的容量可为约0.l cc至约0.7cc,优选容量为约0.2cc至约0.6cc,更优选容量为约0.23cc至约0.5cc,而最优选容量为约0.3cc至约0.4cc。在具体实施方案中,人工授精行业内常把该细长容器称为吸管,其容量为约0.23cc,且长度与直径比例为约133:1。在另一个具体实施方案中,人工授精行业内常把该细长容器称为吸管,其容量为约0.5cc,且长度与直径比为约67:1。一般而言,这些容量的容器可用于贮存牛精子细胞。
或细长容器的容量可为1cc至约100cc,优选容量为约10cc至约75cc,更优选容量为约15cc至约50cc,更优选容量为约20cc至约40cc,甚至更优选容量为约25cc至约30cc。在具体实施方案中,人工授精行业内常把该细长容器称为吸管,其容量为约25cc,且长度与直径比例为约445:1。一般而言,这一容量的容器可用于贮存猪精子细胞。
将精子分散体系贮存于吸管中的优势在于,可一直将分散体系贮存其中直至其被用于授精雌性哺乳动物,彼时即可将吸管内的内容物置入雌性哺乳动物的子宫中。因此,在这种情况下,细胞可保留在吸管的运动性抑制剂内(因此为不运动状态),直至用该分散体系授精雌性,到时子宫内部的液体会稀释运动性抑制剂,使不运动细胞恢复运动。
III.受精或授精
本发明的另一方面主要是利用上述贮存精子的新方法,使卵受精或授精雌性哺乳动物。
一旦如上文“收集精子细胞样品”中详细讨论的,形成了精子分散体系,该精子分散体系即可用于授精雌性哺乳动物。可以根据本领域技术人员已知的多种方法中的任意一种实施授精。这些方法包括如人工授精(包括标准人工授精和深度子宫内授精),以及其他本领域技术人员所熟知的方法。例如,包含不运动精子和诱导精子不运动的组合物的精子分散体系,可通过如人工授精用于授精雌性哺乳动物。在具体实施方案中,该精子分散体系可在细长容器中以用于授精雌性哺乳动物,且该精子分散体系中的精子保持不动直至使用其对雌性哺乳动物授精。在另一实施方案中,通过如上述方法使精子分散体系中的精子恢复了运动或活性状态,然后用于授精雌性哺乳动物。
或者也可通过如微注射(包括卵细胞质内单精子注入(intracytoplasmicsperm injection,ICSI))及其他本领域已知的方法,用该精子分散体系授精卵,更具体而言为体外的卵。然后可通过多种本领域技术人员已知方法中的任意一种,将受精卵导入雌性哺乳动物的子宫内,例如通过胚胎移植。
利用精子分散体系授精雌性哺乳动物或使体外卵细胞受精(随后将受精卵导入雌性子宫中),可在形成精子分散体系之后不久进行,例如在约120小时之内,优选在约96小时之内,更优选在约72小时之内,更优选在约48小时之内,而在具体实施方案中,在形成精子分散体系后的约24小时之内。在这种情况下,一般在授精雌性哺乳动物或使体外卵细胞受精前,该分散体系可不需冷藏(即,分散体系是新鲜的或包含新鲜的精子细胞);或者,可将其保存于运动性抑制剂中,和/或在约4℃至约25℃温度,更优选为约10℃至约25℃,进一步优选为约15℃至约20℃,而最优选为约18℃下冷藏。或者,可冷藏该分散体系,然后在授精雌性哺乳动物或使体外卵细胞受精之前解冻(即,分散体系为冷冻/解冻或包含冷冻/解冻的精子细胞)。一般而言,在这种情况下,将于授精雌性哺乳动物或使体外卵细胞受精之前即刻解冻冷藏的分散体系,例如在约15分钟内。或者可将冷藏的分散体系于一段时间内解冻,或解冻后贮存一段时间,如少于约5天,更优选少于约2天,更优选少于约1天,而最优选少于约12小时。
IV.提供新鲜精子分散体系的方法
本发明的另一个方面主要是利用上述精子分散体系,为授精雌性哺乳动物提供新鲜的精子分散体系。有利的是,运动性抑制剂可以保证从来源哺乳动物精子收集至相应抑制剂中,并将精子分散体系运达育种场所(即,授精雌性哺乳动物的场所)而不需冷冻分散体系。因此,这使得相对于冰冻/解冻分散体系,该分散体系中有更大百分比的精子保持存活,其中冰冻及随后解冻分散体系中细胞的困难增加了对细胞存活力不良影响的风险。运动性抑制剂在精子分散体系中的应用,以及任选于约4℃至约25℃的温度,优选于约10℃至约20℃,更优选于约15℃至约20℃,而最优选为约18℃下贮存和/或运输分散体系,可提高利用该精子分散体系授精雌性哺乳动物的受孕率。运动性抑制剂在精子分散体系中的应用,以及任选在低温下贮存和运输,也使可利用较低精子浓度授精雌性哺乳动物,因为该分散体系中更大数量的细胞保持了存活力。
一旦如上文“收集样品”中详细描述的,形成了精子分散体系,可将该分散体系置于容器中运输到遥远地点,如距离获得精子或形成精子分散体系的场所一或多英里远(1.69或更多千米)的农场或其他育种场所。分散体系可置于任何能保护其免受运输的艰难环境而导致的损伤的容器中。可以将容器隔热以保持分散体系处于合适的温度下,例如在约4℃至约25℃间,更优选约10℃至约25℃间,更优选约15℃至约20℃间,而最优选为约18℃。此外,可将分散体系置于用于授精雌性哺乳动物的容器中,然后将一个或多个该授精容器置于另一容器中运输至遥远地点。例如,可将精子分散体系置于一个或多个吸管中,将这些吸管置于冷却器内运输到育种场所。
可以通过许多已知的运输方法中的任意一种来运输容器,例如通过美国邮递、多种过夜运输服务中的任意一种,或通过快件服务。
上文已经详细描述了本发明,显而易见的是,在不脱离所附权利要求定义的发明范围内可对其进行修饰和改变。
Claims (76)
1.贮存未分选精子的方法,所述方法包括
a.形成包含精子、诱导精子不运动的组合物以及抗生素的精子分散体系,且
b.贮存所述精子分散体系。
2.贮存分选精子的方法,所述方法包括
a.形成包含精子及诱导精子不运动的组合物的精子分散体系,
b.将该精子分散体系分选为分离群体,其中所述群体之一中的精子包含至少约65%带有X染色体的精子细胞或至少约65%带有Y染色体的精子细胞,且
c.于约-4℃至约30℃的温度下贮存上述群体。
3.授精雌性哺乳动物的方法,所述方法包括用精子分散体系授精雌性哺乳动物,所述精子分散体系包含不运动的精子和诱导精子不运动的组合物。
4.提供新鲜精子分散体系用于授精雌性哺乳动物的方法,所述方法包括
a.形成包含精子和诱导精子不运动的组合物的精子分散体系,
b.将该精子分散体系置于容器中用以进行远距离运输,且
c.在形成精子分散体系后的约24小时内将所述容器内的精子分散体系进行远距离运输。
5.一种组合,其包括
a.用于授精雌性哺乳动物的细长容器,及
b.精子分散体系,所述精子分散体系包含不运动的精子和诱导精子不运动的组合物,且其中所述精子分散体系含纳于所述细长容器内。
6.包含不运动的精子、诱导精子不运动的组合物以及冷冻防腐剂的精子分散体系。
7.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系贮存在约-4℃至约30℃。
8.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系贮存在约-4℃至约5℃。
9.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系贮存在约-4℃至约0℃。
10.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系贮存在约0℃至约5℃。
11.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系贮存在约5℃至约30℃。
12.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系贮存至少约24小时。
13.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系贮存至少约48小时。
14.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系贮存至少约72小时。
15.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系贮存至少约96小时。
16.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系贮存至少约1周。
17.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系贮存至少约2周。
18.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系中精子的浓度为至少约0.04×106精子/ml。
19.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系中精子的浓度为至少约1.5×106精子/ml。
20.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系中精子的浓度为至少约2×106精子/ml。
21.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系中精子的浓度为至少约3×106精子/ml。
22.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系中精子的浓度为至少约0.5×107精子/ml。
23.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系中精子的浓度为至少约1×107精子/ml。
24.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系中精子的浓度为至少约2×107精子/ml。
25.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系中精子的浓度为至少约3×107精子/ml。
26.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系中精子的浓度为至少约4×107精子/ml。
27.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系中精子的浓度为至少约5×107精子/ml。
28.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系中精子的浓度为至少约6×107精子/ml。
29.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系中精子的浓度为至少约7×107精子/ml。
30.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系中精子的浓度为至少约8×107精子/ml。
31.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系中精子的浓度为至少约12×107精子/ml。
32.前述任一项权利要求中的方法、组合或分散体系,其中所述诱导精子不运动的组合物包含钾及任选的钠,钾与钠各自的摩尔比大于1:1。
33.前述任一项权利要求中的方法、组合或分散体系,其中诱导精子不运动的组合物包含钾及任选的钠,钾与钠的摩尔比大于1.25:1。
34.前述任一项权利要求中的方法、组合或分散体系,其中诱导精子不运动的组合物包含钾及任选的钠,钾与钠的摩尔比大于1.5:1。
35.前述任一项权利要求中的方法、组合或分散体系,其中诱导精子不运动的组合物包含钾及任选的钠,钾与钠的摩尔比大于1.75:1。
36.前述任一项权利要求中的方法、组合或分散体系,其中诱导精子不运动的组合物包含钾及任选的钠,钾与钠的摩尔比大于2:1。
37.前述任一项权利要求中的方法、组合或分散体系,其中诱导精子不运动的组合物包含钾及任选的钠,钾与钠的摩尔比大于3:1。
38.前述任一项权利要求中的方法、组合或分散体系,其中诱导精子不运动的组合物包含钾及任选的钠,钾与钠的摩尔比大于4:1。
39.前述任一项权利要求中的方法、组合或分散体系,其中诱导精子不运动的组合物包含钾及任选的钠,钾与钠的摩尔比大于5:1。
40.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系中的精子相对于相同种系体内射出的精子,其运动性更具有附睾精子的特征性行为。
41.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系包含碳酸盐来源。
42.前述任一项权利要求中的方法、组合或分散体系,其中所述分散体系包含NaHCO3、KHCO3和C6H8O7·H2O。
43.前述任一项权利要求中的方法、组合或分散体系,其中所述分散体系包含含有0.097摩尔/升NaHCO3、0.173摩尔/升KHCO3、0.090摩尔/升C6H8O7·H2O的水溶液的缓冲液。
44.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系进一步包含冷冻防腐剂。
45.权利要求44中的方法、组合或分散体系,其中所述冷冻防腐剂包含甘油。
46.权利要求44或45中的方法、组合或分散体系,其中所述冷冻防腐剂包含卵黄。
47.权利要求44、45或46中任一项的方法、组合或分散体系,其中所述冷冻防腐剂包含甘油、TRIS、柠檬酸及果糖。
48.权利要求44、45、46或47中任一项的方法、组合或分散体系,其中每升冷冻防腐剂包含60ml甘油、24.2g TRIS、13.8g柠檬酸、10.0g果糖。
49.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系中的精子来自一种以上相同种系的哺乳动物。
50.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系中的精子来自从由牛、猪、马或猪组成的组中选择的哺乳动物。
51.权利要求2或权利要求2的任何从属权利要求的方法,其中群体之一的精子包含至少约70%带有X染色体的精子细胞或至少约70%带有Y染色体的精子细胞。
52.权利要求2或权利要求2的任何从属权利要求的方法,其中群体之一的精子包含至少约75%带有X染色体的精子细胞或至少约75%带有Y染色体的精子细胞。
53.权利要求2或权利要求2的任何从属权利要求的方法,其中群体之一的精子包含至少约80%带有X染色体的精子细胞或至少约80%带有Y染色体的精子细胞。
54.权利要求2或权利要求2的任何从属权利要求的方法,其中群体之一的精子包含至少约85%带有X染色体的精子细胞或至少约85%带有Y染色体的精子细胞。
55.权利要求2或权利要求2的任何从属权利要求的方法,其中群体之一的精子包含至少约90%带有X染色体的精子细胞或至少约90%带有Y染色体的精子细胞。
56.权利要求2或权利要求2的任何从属权利要求的方法,其中群体之一的精子包含至少约95%带有X染色体的精子细胞或至少约95%带有Y染色体的精子细胞。
57.权利要求3或4的方法、或权利要求5的组合、或权利要求3、4或5的任何从属权利要求中的方法或组合,其中所述精子分散体系包含分选为分离群体的精子,其中群体之一的精子包含至少约65%带有X染色体的精子细胞或至少约65%带有Y染色体的精子细胞。
58.权利要求3或4的方法、或权利要求5的组合、或权利要求3、4或5的任何从属权利要求中的方法或组合,其中所述精子分散体系包含分选为分离群体的精子,其中群体之一的精子包含至少约70%带有X染色体的精子细胞或至少约70%带有Y染色体的精子细胞。
59.权利要求3或4的方法、或权利要求5的组合、或权利要求3、4或5的任何从属权利要求中的方法或组合,其中所述精子分散体系包含分选为分离群体的精子,其中群体之一的精子包含至少约75%带有X染色体的精子细胞或至少约75%带有Y染色体的精子细胞。
60.权利要求3或4的方法、或权利要求5的组合、或权利要求3、4或5的任何从属权利要求中的方法或组合,其中所述精子分散体系包含分选为分离群体的精子,其中群体之一的精子包含至少约80%带有X染色体的精子细胞或至少约80%带有Y染色体的精子细胞。
61.权利要求3或4的方法、或权利要求5的组合、或权利要求3、4或5的任何从属权利要求中的方法或组合,其中所述精子分散体系包含分选为分离群体的精子,其中群体之一的精子包含至少约85%带有X染色体的精子细胞或至少约85%带有Y染色体的精子细胞。
62.权利要求3或4的方法、或权利要求5的组合、或权利要求3、4或5的任何从属权利要求中的方法或组合,其中所述精子分散体系包含分选为分离群体的精子,其中群体之一的精子包含至少约90%带有X染色体的精子细胞或至少约90%带有Y染色体的精子细胞。
63.权利要求3或4的方法、或权利要求5的组合、或权利要求3、4或5的任何从属权利要求中的方法或组合,其中所述精子分散体系包含分选为分离群体的精子,其中群体之一的精子包含至少约95%带有X染色体的精子细胞或至少约95%带有Y染色体的精子细胞。
64.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系是冷冻的。
65.权利要求1-63中任一项的方法、组合或分散体系,其中所述精子分散体系是未冷冻的。
66.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系在用于授精雌性哺乳动物之前贮存了至少约24小时。
67.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系在用于授精雌性哺乳动物之前贮存了至少约48小时。
68.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系在用于授精雌性哺乳动物之前贮存了至少约72小时。
69.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系在用于授精雌性哺乳动物之前贮存了至少约96小时。
70.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系在用于授精雌性哺乳动物之前贮存了至少约120小时。
71.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系在形成之后的约24小时之内运输。
72.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系在形成之后的约48小时之内运输。
73.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系在形成之后的约72小时之内运输。
74.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系在形成之后的约96小时之内运输。
75.前述任一项权利要求中的方法、组合或分散体系,其中所述精子分散体系在形成之后的约120小时之内运输。
76.权利要求5或任何权利要求5的从属权利要求中的组合,其中所述容器为人工授精的吸管。
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