CN117562046A - 一种羊精液冻存液及其应用 - Google Patents
一种羊精液冻存液及其应用 Download PDFInfo
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- CN117562046A CN117562046A CN202311416916.0A CN202311416916A CN117562046A CN 117562046 A CN117562046 A CN 117562046A CN 202311416916 A CN202311416916 A CN 202311416916A CN 117562046 A CN117562046 A CN 117562046A
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- Prior art keywords
- semen
- sheep
- gsh
- solution
- diluent
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Classifications
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- 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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- 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
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- 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
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
本发明提供了一种羊精液冻存液及其应用,将GSH‑Px用于羊精液的保存中。本发明还提供的羊精液冻存液,包括GSH‑Px,还包括GSH和MitoQ。本发明将GSH‑Px用于羊精液的保存,提高了保存后羊精液的活力以及存活时间,提升了羊精液品质。GSH‑Px、GSH和MitoQ共同用于羊精液的保存,羊精液品质更好,受胎率更高;经试验证实,本发明提供的精液冻存液能够显著提高精液冷冻保存活性,延长精子体外保存时间。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种羊精液冻存液及其应用。
背景技术
随着家畜养殖业向集约化、规模化发展,人工授精技术也逐渐被广泛应用。人工授精技术是高效扩繁的重要技术手段,具有提高配种率,减少公畜饲养量和疾病传播,降低引种费用,显著提高经济效益,便于集中统一管理等诸多优点,而精液保存是人工授精技术的关键环节,对于提高优种公畜的利用率,推进家畜的遗传改良工作有重要意义。
精液在冷冻保存过程中会诱导精子功能的有害变化,包括ROS的产生,其促进膜脂质的过氧化,降低精子活力和线粒体活性,并增加DNA片段化和氧化。过度的ROS会导致精子功能缺陷,包括膜损伤、DNA碎裂和线粒体功能障碍,然后最终导致精子运动和受精潜能受损。为此,各种外源性抗氧化剂已广泛补充到公羊冷冻延长剂中,以消除ROS造成的有害影响。在稀释液中补充外源性抗氧化剂以改善氧化应激和改善解冻后精液质量,可以减轻精液在冷冻保存过程中的过氧化损伤,延长精液保存期。
Eslami等研究了公羊精液的液态保存,发现棕榈油酸可以作为一种抗氧化剂来保护精子免受损伤,用它处理72h可以显著提高精子的活力和运动能力,并且通过PCR测定进一步证明榈油酸可以提高精子的抗氧化水平及超氧化物歧化酶(SOD)活性。
在过去的几十年中,冷冻精液技术迅猛发展,不同动物的精液冷冻都获得了较快的进展。羊的精子与其他家畜相比具有一定生理特性,冷冻后活力低、受胎率低,且不稳定,成为制约羊冷冻精子发展的桎梏。
发明内容
本发明提供一种羊精液冻存液及其应用,用以解决现有技术中绵羊的精子冷冻后活力低、受胎率低,且不稳定的缺陷,本发明将GSH-Px用于羊精液保存,提高了保存后羊精液的活力以及存活时间,提升羊精液品质。
本发明提供了GSH-Px在羊精液保存中的应用。
GSH-Px(Glutathione peroxidase,谷胱甘肽过氧化物酶)是机体内广泛存在的一种重要的过氧化物分解酶,也是体内重要的自由基捕获酶之一。GSH-Px不仅具有清除自由基和衍生物的作用,还减少脂质过氧化物的形成,增强机体抗氧化损伤的能力。GSH-Px还参与前列腺素和血栓素的合成,保护细胞的结构和功能。此外,GSH-Px能够通过降低线粒体氧化损伤保护线粒体,从而降低细胞氧化应激水平,提高生殖细胞冷冻后活力。
精子呼吸的耗氧量通常按109个精子在37℃下所消耗的氧量计算,正常牛、鸡、兔和绵羊精子的耗氧量分别为21、7、11和22μL。可见,绵羊精子的耗氧量较高。
本发明将GSH-Px(谷胱甘肽过氧化物酶)作为一种外源性抗氧化剂用于羊精液保存,可以破坏过氧化物的作用,增强精子的抗氧化能力,避免氧化应激损伤,延长精子的保存期,提升绵羊冷冻精液品质,提高绵羊冷冻精液解冻后活力以及存活时间。
在一些实施例中,所述精液为绵羊精液;
优选地,所述保存为冷冻保存;
更优选地,将GSH-Px的稀释液用于绵羊精液的冷冻保存。
冷冻可以使精子的代谢活动受到抑制,当温度恢复时,仍能保持活力。但冻融过程可产生大量的ROS,因精子质膜富含多聚不饱和脂肪酸,易受氧化应激损伤。绵羊的精子冷冻后解冻测定发现活力较低、受胎率低,且不稳定。
本发明采将GSH-Px的稀释液用于绵羊精液的冷冻保存,可明显改善冷冻保存的绵羊精液品质;同时提高绵羊精子的运动能力,提高精子质膜完整性和线粒体活性。
本发明还提供了一种羊精液冻存液。
所述羊精液冻存液中,GSH-Px的添加量为0.01-100U/mL;
优选地,所述羊精液冻存液中,GSH-Px的添加量为25-100U/mL;
更优选地,GSH-Px的添加量为50-100U/mL。
虽然过量的ROS会导致精子功能缺陷,但在生理水平产生的ROS是获能、顶体反应、超活化和获得受精能力所必需的。本发明在羊精液冻存液中,添加一定浓度的GSH-Px,可以破坏过氧化物,但过量的GSH-Px会造成ROS的过量清除,为维持细胞内正常生理水平的ROS,在羊精液冻存液中,添加25-100U/mL,优选50-100U/mL,进一步优选50U/mL的GSH-Px对羊精液进行冷冻保存,可显著提高绵羊精子冻后质量,包括冻后活力,运动能力都显著提升,维持质膜和DNA完整,保护顶体和线粒体不受损伤。在一些实施例中,所述羊精液冻存液中还包括甘油、抗冻剂、GSH和MitoQ;
本发明在羊精液冻存液中还添加了甘油和抗冻剂,两者对精子具有防冻保护作用,可以提高精子的复苏率。添加的GSH和MitoQ是为了与GSH-Px配合共同起到清除ROS,提高精子抗氧化损伤能力的作用。
谷胱甘肽(GSH)是哺乳动物细胞中主要的非蛋白巯基化合物,具有多种生物学功能,是精液本身存在的抗氧化剂,可以快速地被细胞用作氧化应激的防御。谷胱甘肽的巯基(SH)赋予其抗氧化损伤保护作用。GSH的缺乏会导致精子中段的不稳定,从而导致运动障碍。GSH保护细胞膜脂质氧化,消除超级过氧化物和阻止氧化。GSH可以清除脂质过氧化物,从而抑制过氧化连锁反应。GSH也可以清除H2O2,避免脂质过氧化。
MitoQ(线粒体靶向抗氧化剂)能通过降低ROS水平,减少线粒体蛋白、脂质和DNA氧化损伤而保护线粒体。本发明在羊精液冻存液中添加MitoQ精子前向运动和总活动率都有所增加。MitoQ与GSH和GSH-Px共用,可以有效减少ROS,冷冻复苏后精子活力明显增加,能有效改善精子生育潜力。
在一些实施例中,每100mL所述羊精液冻存液中,甘油4-8mL,抗冻剂1-3mL,GSH4.5-5.5mM,MitoQ 5-10μM;
优选地,所述羊精液冻存液中还包括鸡蛋卵黄稀释液;更优选地,以体积百分含量计,所述鸡蛋卵黄稀释液中鸡蛋卵黄液的添加量为10-20%。
本发明在羊精液冻存液中还添加了鸡蛋卵黄,蛋黄中含有大量的维生素、矿物质及高生物价值的蛋白质,为精子提供充足的营养。
进一步优选地,所述鸡蛋卵黄稀释液中还包括基础稀释液,所述基础稀释液包括三羟甲基氨基甲烷、果糖、柠檬酸和青链霉素。
所述基础稀释液中,按重量份计,三羟甲基氨基甲烷1-2份,果糖0.3-0.8份,柠檬酸1.6-2.0份;青链霉素的添加量为90-110IU/100mL,余量为水。
其中,三羟甲基氨基甲烷为缓冲液,果糖为精子在体外代谢提供能量来源,柠檬酸调节基础稀释液的pH;青链霉素为抗生素,具有抑菌效果。三羟甲基胺基甲烷、柠檬酸及果糖共用可维持稀释液环境与精子内环境的渗透压平衡。
本发明还提供了一种用于羊精液保存的试剂盒,所述试剂盒包括所述的精液冻存液;优选地,所述试剂盒包括基础稀释液、鸡蛋卵黄稀释液和/或包括GSH-Px的混合液;更优选地,包括GSH-Px的混合液为甘油,抗冻剂,GSH,MitoQ,GSH-Px组成的混合液。
本发明还提供了一种羊精液冷冻保存的方法。
羊精液冷冻保存的方法包括:将所述的精液冻存液与精液混合后进行冻存;
优选地,所述精液冻存液与精液按体积比为(2-7):1进行混合。
在一些实施例中,所述冻存为先冷藏再冷冻;
优选地,所述冷藏的温度为0-4℃;
更优选地,先将精液冻存液与精液的混合液降温到0-4℃,保温1-2小时,再进行冷冻;
进一步优选地,所述冷冻的温度为-18℃以下。
使用时,羊精液冻存前,先将采集的羊精液与预热至37℃左右的基础稀释液按体积比为1:0.5混合进行稀释,迅速检测精子活力与密度,活率0.8以上,精子密度2×108个/mL以上方可使用,否则弃去。然后将羊精液与鸡蛋卵黄稀释液(Ⅰ液)等体积混合放入冰箱降温2h,从37℃降温到0-4℃,再将羊精液与甘油4-8mL,抗冻剂1-3mL,GSH 4.5-5.5mM,MitoQ5-10μM与GSH-Px组成的Ⅱ液体积比1:2.5混合后放入0-4℃冰箱中平衡1.5h,最后,将平衡后的混合液置于冻精细管中,于液氮中冷冻保存。
精液加入精液冻存液,经冷冻后还需解冻才能使用。
在一些实施例中,冻存后解冻的方法包括:将冻存液(羊精液与II液的混合液)在35-39℃预热,再与35-39℃的稀释液混合备用;
优选地,预热的时间为20-40s;
更优选地,所述稀释液为基础稀释液;所述基础稀释液中,按重量份计,三羟甲基氨基甲烷1-2份,果糖0.3-0.8份,柠檬酸1.6-2.0份;青链霉素的添加量为90-110IU/100mL,余量为水。
本发明将GSH-Px用于羊精液的保存,提高了保存后羊精液的活力以及存活时间,提升了羊精液品质。经试验证实,本发明提供的精液冻存液能够显著提高精液冷冻保存活性,延长精子体外保存时间;同时,冷冻解冻后精子活力与不添加GSH-Px相比能提高15%左右。
本发明采用GSH-Px、GSH和MitoQ共用得到的精液冻存液,用于羊精液的保存,羊精液品质更好,受胎率更高;本发明的精液冻存液使用方便,操作简便,应用成本低,效果显著,对羊不产生任何有害影响。可以为羊精液保存提供一定的数据支撑,尤其是在绵羊精液保存及相关的胚胎移植产业(人工授精技术)和胚胎工程中广泛应用,具有广阔的市场前景。
附图说明
为了更清楚地说明本发明或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明试验例1不同GSH-Px添加量的羊精液冻存液对精子冷冻保存活率的影响;
图2为本发明试验例1不同GSH-Px添加量的羊精液冻存液对精子冷冻保存活力的影响;
图3为本发明试验例1不同GSH-Px添加量的羊精液冻存液对精子冷冻保存DNA完整性的影响;
图4为本发明试验例1不同GSH-Px添加量的羊精液冻存液对精子冷冻保存质膜完整性的影响;
图5为本发明试验例1不同GSH-Px添加量的羊精液冻存液对精子冷冻保存线粒体活性的影响;
图6为本发明试验例1不同GSH-Px添加量的羊精液冻存液对精子冷冻保存顶体完整性的影响;
图7为本发明试验例1中分别添加0U/mL、50U/mL GSH-Px的羊精液冻存液对绵羊人工授精妊娠率的影响。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合本发明中的附图,对本发明中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
下面结合图1-图7描述本发明的GSH-Px在羊精液保存中的应用。
在一些实施例中,将GSH-Px在羊精液保存中应用;
优选地,所述精液为绵羊精液;
更优选地,将GSH-Px的稀释液用于绵羊精液的冷冻保存。
在一些实施例中,所述羊精液冻存液中,GSH-Px的添加量为0.01-100U/mL;
优选地,所述羊精液冻存液中,GSH-Px的添加量为25-100U/mL;
更优选地,GSH-Px的添加量为50-100U/mL。
在一些实施例中,所述羊精液冻存液中还包括甘油、抗冻剂、GSH和MitoQ;
在一些实施例中,每100mL所述羊精液冻存液中,甘油4-8mL,抗冻剂1-3mL,GSH4.5-5.5mM,MitoQ 5-10μM;
在一些实施例中,所述抗冻剂采购自北京田园奥瑞生物科技有限公司。
优选地,所述羊精液冻存液中还包括鸡蛋卵黄稀释液;更优选地,以体积百分含量计,所述鸡蛋卵黄稀释液中鸡蛋卵黄液的添加量为10-20%。
进一步优选地,所述鸡蛋卵黄稀释液中还包括基础稀释液,所述基础稀释液包括三羟甲基氨基甲烷、果糖、柠檬酸和青链霉素。
所述基础稀释液中,按重量份计,三羟甲基氨基甲烷1-2份,果糖0.3-0.8份,柠檬酸1.6-2.0份;青链霉素的添加量为90-110IU/100mL,余量为水。
在一些实施例中,一种用于羊精液保存的试剂盒,所述试剂盒包括所述的精液冻存液。
在一些实施例中,羊精液冷冻保存的方法包括:将所述的精液冻存液与精液混合后进行冻存;
优选地,所述精液冻存液与精液按体积比为(2-7):1进行混合。
在一些实施例中,所述冻存为先冷藏再冷冻;
优选地,所述冷藏的温度为0-4℃;
更优选地,先将精液冻存液与精液的混合液降温到0-4℃,保温1-2小时,再进行冷冻;
进一步优选地,所述冷冻的温度为-18℃以下。
试验中GSH-Px、GSH和MitoQ均购自Sigma-Aldrich公司,本发明中的其他试剂除特殊说明外,均为市售常规产品。
试验例1
本试验例提供一种羊精液冻存液及提高羊精液冷冻保存品质的方法,包括以下步骤:
(1)提取绵羊的新鲜精液,并将提取的新鲜精液放入5ml离心管中。
(2)将步骤(1)中盛有精液运送至实验室,运送过程中防止剧烈震荡。
(3)准备制作精液冻存液的原材料,三羟甲基氨基甲烷、果糖、柠檬酸、鸡蛋卵黄、甘油、抗冻剂、青链霉素、GSH、MitoQ、GSH-Px。
(4)采用步骤(3)中的原材料配制基础稀释液,鸡蛋卵黄稀释液(Ⅰ液)和精液冻存液(Ⅱ液);
基础稀释液以100mL计,称取三羟甲基氨基甲烷1.3g,果糖0.56g,柠檬酸1.8g,100IU青链霉素;加入蒸馏水,用磁力搅拌器搅拌均匀,充分溶解后加入容量瓶中,定容到100mL,摇匀后4℃保存;
Ⅰ液以100mL计,取15mL鸡蛋卵黄液,与基础稀释液充分混匀后定容至100mL,并使用微型漩涡混合仪充分混匀,现配现用;
Ⅱ液以100mL计,向容量瓶中加入甘油6mL,抗冻剂2mL,GSH 5mM,MitoQ 5-10μM,GSH-Px 0-100U/mL,充分混匀后加入Ⅰ液并定容至100mL,并使用微型漩涡混合仪充分混匀,现配现用。
(5)将步骤(4)Ⅱ液的制备过程中,GSH-Px分别按照四种不同的添加量(100U/mL、50U/mL、25U/mL、0),具体为:
Ⅱ液A(对照组1未添加GSH-Px):向容量瓶中加入甘油6mL,抗冻剂2mL,GSH 5mM,MitoQ 5-10μM,充分混匀后加入Ⅰ液并定容至100mL,并使用微型漩涡混合仪充分混匀。
Ⅱ液B:向容量瓶中加入甘油6mL,抗冻剂2mL,GSH 5mM,MitoQ 5-10μM,GSH-Px25U/mL,充分混匀后加入Ⅰ液并定容至100mL,并使用微型漩涡混合仪充分混匀。
Ⅱ液C:向容量瓶中加入甘油6mL,抗冻剂2mL,GSH 5mM,MitoQ 5-10μM,GSH-Px50U/mL,充分混匀后加入Ⅰ液并定容至100mL,并使用微型漩涡混合仪充分混匀。
Ⅱ液D:向容量瓶中加入甘油6mL,抗冻剂2mL,GSH 5mM,MitoQ 5-10μM,GSH-Px100U/mL,充分混匀后加入Ⅰ液并定容至100mL,并使用微型漩涡混合仪充分混匀。
Ⅱ液E(对照组2未添加MitoQ):容量瓶中加入甘油6mL,抗冻剂2mL,GSH 5mM,GSH-Px 50U/mL,充分混匀后加入Ⅰ液并定容至100mL,并使用微型漩涡混合仪充分混匀。
Ⅱ液F(对照组3未添加GSH):容量瓶中加入甘油6mL,抗冻剂2mL,MitoQ 5-10μM,GSH-Px 50U/mL,充分混匀后加入Ⅰ液并定容至100mL,并使用微型漩涡混合仪充分混匀。
Ⅱ液G(对照组4未添加GSH、MitoQ和GSH-Px):容量瓶中加入甘油6mL,抗冻剂2mL,充分混匀后加入Ⅰ液并定容至100mL,并使用微型漩涡混合仪充分混匀。
(6)对步骤(2)中的鲜精进行稀释:采精前将基础稀释液置于37℃水浴锅内进行预热,将采集的绵羊精液与预热至37℃的基础稀释液按体积比为1:0.5混合进行稀释,迅速检测精子活力与密度,活率0.8以上,精子密度2×108个/mL以上方可使用,否则弃去。
然后将符合要求的绵羊精液与Ⅰ液等体积混合放入冰箱降温2h,从37℃降温到4℃;
另取符合要求的绵羊精液,与步骤(5)中不同浓度的Ⅱ液(Ⅱ液A、Ⅱ液B、Ⅱ液C、Ⅱ液D分别以体积比1:2.5混合后放入4℃冰箱中平衡1.5h。
(7)对步骤(6)中稀释的精液分别冷冻保存:精液平衡结束后在低温操作柜中轻轻混匀后用注射器将精液吸入冻精细管,封口,将封好的细管摆放在托架上,随后将托架放入程序冷冻仪器,对精液进行冷冻。精液冷冻完成后,将细管装入布袋做好标记保存在液氮罐中。
(8)对步骤(7)中的精液进行解冻:将布袋从液氮罐里取出置于装有液氮的泡沫盒内,用长柄镊子夹出细管迅速放入37℃水浴锅内轻轻摇晃30s后取出,用无菌纱布将细管上的水分擦干,随后用专用剪刀剪去封口端,将剪开的一端迅速放入37℃内预热好的离心管中,使其缓缓流入离心管。
试验效果检测
1、对步骤(8)中解冻的4组稀释精液的活力、活率、DNA完整性、质膜完整性、线粒体活性、顶体完整性进行检测,具体如下:
(1)活力、活率的测定
将解冻后的精液放入预热离心管中后再加入三倍体积的基础稀释液,轻轻摇晃混匀,在37℃水浴锅中孵育3min。用移液枪吸取10μL精液滴于预热好的载玻片上,轻轻覆盖盖玻片,将载玻片置于400倍显微镜下。通过精子辅助分析仪(CASA)分析,每次至少随机检测5个视野。
运动的精子占精子总数的比例即为精子活力,精子中呈直线运动的精子数占精子总数的百分比即为精子活率,然后依次对Ⅱ液A、B、C、D、的精子活率、活力进行测定,比较GSH-Px对精子冷冻保存活率、活力的影响,结果如图1和图2所示。
(2)DNA完整性测定
使用吖啶橙与碘化乙啶(EB)双标荧光染色法,向提前预热好的离心管中加入95μL稀释后的精液,迅速加入3μL吖啶橙及4μL EB,快速混匀,37℃避光孵育15min,然后加入8μL的Hancock’s溶液吹打混匀终止反应,使用移液枪吸取8μL的混合液于载玻片上,轻轻覆盖盖玻片,压片时尽量避免气泡的产生,使精子不再流动。将载玻片置于400倍荧光显微镜下观察,随机选取视野,快速拍照,计数至少400个精子。
吖啶橙/EB双标荧光染色的结果为:精子头部呈绿色为DNA双链精子,红色为DNA单链精子,橙黄色为DNA双链不稳定精子。精子DNA完整性为DNA双链精子数占精子总数的百分比,然后依次对Ⅱ液A、B、C、D的精子DNA完整性进行测定,结果如图3所示。
(3)质膜完整性测定
使用低渗膨胀法(HOST)向提前预热好的离心管中加入20μL精液,随后向离心管中加入180μL预热好的低渗溶液(逐步加入),轻轻摇晃混匀,在37℃条件下孵育60min。使用移液枪吸取8μL混合液于载玻片上,涂片,自然风干后置于400×视野下拍照,随机选取视野,计数至少400个精子。弯尾参照世界卫生组织(WHO)制定的精子尾部肿胀标准。
精子质膜完整率为质膜完整的精子占总精子数的百分比,然后依次对Ⅱ液A、B、C、D的精子质膜完整性进行测定,结果如图4所示。
(4)线粒体活性测定
使用JC-1与碘化丙啶(PI)双标荧光染色法,向提前预热好的离心管中加入55μL稀释后的精液,迅速加入2μL JC-1及8μL PI,快速混匀,37℃避光孵育15min,然后加入8μL的Hancock’s溶液吹打混匀终止反应。使用移液枪吸取4μL的混合液于载玻片上,轻轻覆盖盖玻片,手指轻轻按压盖玻片挤出气泡及多余的液体,使精子不再流动。将载玻片置于400倍荧光显微镜下观察,随机选取视野,快速拍照,计数至少400个精子。
JC-1/PI双荧光染色的结果为:尾部呈橙色荧光的精子为高线粒体膜电位精子;尾部呈绿色荧光的精子为低线粒体膜电位精子。精子线粒体活性为高线粒体活性的精子占精子总数的百分比,然后依次对Ⅱ液A、B、C、D的精子线粒体活性进行测定,结果如图5所示。
(5)顶体完整性测定
使用金霉素荧光染色法(CTC):向提前预热好的离心管中加入30μL稀释后的精液,然后加入等量CTC染色液,快速混匀,37℃避光孵育1min,然后加入8μL的Hancock’s溶液吹打混匀终止反应,使用移液枪吸取4μL的混合液于载玻片上,轻轻覆盖盖玻片,压片时尽量避免气泡的产生,使精子不再流动。将载玻片置于400倍荧光显微镜下观察,随机选取视野,快速拍照,计数至少400个精子。
金霉素荧光染色结果为:精子头部发出强烈黄绿色荧光为顶体完整精子,精子头部无荧光为顶体损伤精子。精子顶体完整性为顶体完整精子数占精子总数的百分比,然后依次对Ⅱ液A、B、C、D的精子顶体完整性进行测定,结果如图6所示。
(6)抗氧化相关指标测定
抗氧化酶的提取与保存:取120μL解冻后精液样品25℃1600×g离心10min,弃去上清,向沉淀中加入400μL 1% TritonX-100,冰上裂解20min,裂解完成后将样品以25℃10000×g离心15min,保留上清液,分装后-80℃冷冻保存备用。
MDA使用南京建成MDA试剂盒(货号A003-1,TBA法)进行测定,照着说明书依次加入待测样品和相应试剂后,95℃水浴80min,取出后流水冷却4000转/分,离心10min。吸取上清200μL,使用酶标仪在532nm处,1cm光径,测定各管吸光度以计算MDA含量,结果以nmoL/mL为单位。
T-AOC使用南京建成T-AOC试剂盒(货号A015-2-1,ABTS法)进行测定,制作标准曲线,使用作图工具制得曲线公式。按照操作步骤添加待测样品及试剂,室温反应6min,波长405nm处读取OD值,将测得OD值代入曲线公式求得结果,结果以mM为单位。
SOD、CAT、GSH-Px均用试剂盒提供的方法测定,试剂盒均购自南京建成生物工程研究所,根据试剂盒的使用方法分别检测解冻后各试验组的酶活性,结果如表1所示。
表1解冻后各试验组的酶活性
注:不同肩标字母表示差异显著。
从图1~图6可知,对照试验即添加II液A的试验在绵羊冷冻保存稀释液中未加入GSH-Px,而II液B、II液C、II液D中加入了GSH-Px,且GSH-Px添加的量分别为25U/mL、50U/mL、100U/mL,结果显示:II液C的添加(GSH-Px的添加量为50U/mL)可显著提高绵羊精子冻后质量,包括冻后活力、运动能力显著提升,维持质膜和DNA完整,保护顶体和线粒体不受损伤;从表1中可以看出,GSH-Px的添加有效的降低了MDA浓度和ROS水平,显著提高了解冻后绵羊精子SOD活性、T-AOC、GSH-Px及CAT活性,II液C添加(GSH-Px的添加量为50U/mL)时,抗氧化效果最好。
2、受胎率的测定
根据上述效果检测试验的检测结果选取抗氧化性最好的一组(II液C)与不添加GSH-Px的II液A组进行体外人工授精并检查受胎率。
选择153只身体健康,体态适中,体重在38~42kg的适龄绵羊母羊,埋置CIDR栓,在第14天撤栓(放栓当天为第1天),撤栓的同时注射330单位孕马血清促性腺激素。在撤栓第3天下午(撤栓当天为第1天)取保存的冷冻精液,置于37℃水浴锅中解冻备用。输精方法为子宫颈口输精。输精时,实验羊头低尾高保定,用温生理盐水清洗并擦拭外阴部,通过开膣器打开阴道,使用头灯观察并定位子宫颈口,利用输精器吸取0.3mL精液,并缓慢注入子宫颈口内。12h后进行第2次输精,24h后进行第3次输精。妊娠检查在输精后第45天,使用B超仪诊断母羊妊娠情况,统计怀孕母羊,并计算受胎率,结果如图7所示。
结果显示,采用II液C组绵羊精液冻存液冷冻精液输精后的母羊妊娠率明显高于II液A组。即在精液冷冻稀释液中添加50U/mL的GSH-Px可明显提高母羊妊娠率。
上述试验中:
步骤(1)中提取精液的对象为绵羊,新鲜精液中的精子存活率在0.8以上,防止提取的绵羊精液中的精子存活率较低而影响后期实验的结果。
步骤(2)中水浴锅的温度为37℃,由于绵羊正常体温在37℃左右,可使提取后精子的温度与绵羊正常体温保持一致,增加绵羊精子的存活率,从而防止水浴锅温度过高或者过低造成绵羊精子大量死亡。
步骤(2)中运输时间不能超过30min,提取后的绵羊鲜精液中的精子,由于运输过程中恒温箱的温度为37℃,正常温度下绵羊鲜精液中的精子存活时间较短,会影响后期的实验结果。
步骤(6)中加稀释液时沿着离心管的管壁将稀释液加入精液,边加边混匀,避免分步稀释和快速稀释,防止稀释对精子造成的不可逆恢复。
由以上实验结果可得,GSH-Px作为一种抗氧化剂能提高绵羊精子质量,推测GSH-Px可以穿过线粒体双层膜,达到线粒体内部发挥清除ROS的作用,从而减弱脂质过氧化反应,降低精子在冷冻过程中因氧化应激而受到的损伤,因此,GSH-Px对精子的保护作用可能是通过降低线粒体氧化损伤保护线粒体,从而降低细胞氧化应激水平,提高生殖细胞冷冻后活力。
本发明相较于传统精液冷冻保存的方法,拥有成本低、操作简易等优点。并且,在绵羊冷冻保存稀释液中添加GSH-Px可迅速作用于精子,有着极强的抗氧化能力,能保护精子避免氧化应激损伤,进而提高精液冷冻保存效果。
最后应说明的是:以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (9)
1.GSH-Px在羊精液保存中的应用,其特征在于,将GSH-Px添加在羊精液中,用于延长羊精液的保存期限。
2.根据权利要求1所述的应用,其特征在于,所述羊精液为绵羊精液;
优选地,所述保存为冷冻保存;
更优选地,将GSH-Px的稀释液用于绵羊精液的冷冻保存。
3.一种羊精液冻存液,其特征在于,所述羊精液冻存液中,GSH-Px的添加量为0.01-100U/mL;
优选地,所述羊精液冻存液中,GSH-Px的添加量为25-100U/mL。
4.根据权利要求3所述的羊精液冻存液,其特征在于,所述羊精液冻存液中还包括GSH和MitoQ;
优选地,所述羊精液冻存液中还包括甘油和抗冻剂;
更优选地,所述羊精液冻存液中还包括鸡蛋卵黄稀释液;以体积百分含量计,所述鸡蛋卵黄稀释液中鸡蛋卵黄液的添加量为10-20%;
进一步优选地,所述鸡蛋卵黄稀释液中还包括基础稀释液,所述基础稀释液包括三羟甲基氨基甲烷、果糖、柠檬酸和青链霉素。
5.根据权利要求4所述的羊精液冻存液,其特征在于,每100mL所述羊精液冻存液中,甘油4-8mL,抗冻剂1-3mL,GSH 4.5-5.5mM,MitoQ 5-10μM;
所述基础稀释液中,按重量份计,三羟甲基氨基甲烷1-2份,果糖0.3-0.8份,柠檬酸1.6-2.0份;青链霉素的添加量为90-110IU/100mL,余量为水。
6.一种用于羊精液保存的试剂盒,其特征在于,所述试剂盒包括权利要求3-5任一所述的精液冻存液。
7.一种羊精液冷冻保存的方法,其特征在于,所述方法包括:将权利要求3-5任一所述的精液冻存液与精液混合后进行冻存;
优选地,所述精液冻存液与精液按体积比为(2-7):1进行混合。
8.根据权利要求7所述的羊精液冷冻保存的方法,其特征在于,所述冻存为先冷藏再冷冻;
优选地,所述冷藏的温度为0-4℃;
更优选地,先将精液冻存液与精液的混合液降温到0-4℃,保温1-2小时,再进行冷冻;
进一步优选地,所述冷冻的温度为-18℃以下。
9.根据权利要求7或8所述的羊精液冷冻保存的方法,其特征在于,冻存后解冻的方法包括:将冻存液在35-39℃预热,再与35-39℃的稀释液混合备用;
优选地,预热的时间为20-40s;
更优选地,所述稀释液为基础稀释液;所述基础稀释液中,按重量份计,三羟甲基氨基甲烷1-2份,果糖0.3-0.8份,柠檬酸1.6-2.0份;青链霉素的添加量为90-110IU/100mL,余量为水。
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