CN109554337B - 透明质酸的用途 - Google Patents
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Abstract
一种透明质酸的新用途,属于动物繁殖技术领域,用于降低精子离心损伤。本发明的优点是:通过添加透明质酸,降低了离心对精子产生的物理损伤,同时不影响精子活力的方法。
Description
技术领域
本发明属于动物繁殖技术领域,具体涉及一种在精子离心时添加透明质酸降低离心过程对精子顶体与膜损伤的方法。
背景技术
目前,家畜种质资源日益重要,为了精液的进一步应用,通常精液在采集后或解冻后经常需要进行洗涤然后离心,而离心过程对精子会产生不可避免的物理损伤。不同品种的家畜由于精子大小等不同,精子离心损伤的程度也会不同,尤其是一些动物的冷冻精子,解冻后再离心可能会严重破坏精子的质膜与顶体,甚至影响受精。
发明内容
本发明解决的问题是离心对精子产生物理损伤的问题,提供一种添加透明质酸降低精子离心损伤的方法使离心过程对精子膜与顶体的损伤降低,提升精子的受胎能力。
本发明的目的是通过以下技术方案实现的;一种透明质酸的用途,其特征在于:用于降低精子离心损伤。
所述降低精子离心损伤是指降低离心对精子膜与顶体的损伤。
所述透明质酸的用途,其特征在于:用于降低精子离心损伤的方法包括以下步骤:
(1)、透明质酸的配制
透明质酸溶液:每毫升精子洗涤液添加0.5mg~4mg的透明质酸,,37度放置5min-10min,轻轻上下颠倒使透明质酸混匀;
(2)、将鲜精或冻精放入加入透明质酸的精子洗涤液中轻轻混匀后离心;
(3)、离心后吸去上清,将精子沉淀重悬。
本发明的优点和有益效果是:
通过添加透明质酸,降低了离心对精子产生的物理损伤,同时不影响精子活力的方法。
具体实施方式
实施例1:
1、透明质酸的配制
0.5mg/ml透明质酸溶液的配制:称取0.5mg透明质酸(国产)加入1ml的PBS中,37度5min,轻轻上下颠倒将液体混匀;
2mg/ml透明质酸溶液的配制:称取2mg透明质酸(国产)加入1ml的PBS中,37度5min,轻轻上下颠倒将液体混匀;
4mg/ml透明质酸溶液的配制:称取4mg透明质酸(国产)加入1ml的PBS中,37度5min,轻轻上下颠倒将液体混匀;
2、取4只冷冻羊精液,解冻后平均分为5组,每份100μl;A组:加入1mlPBS,不离心;B组:加入1mlPBS,离心;C组:加入1mlPBS,含0.5mg/ml透明质酸,离心;D组:加入1mlPBS,含2mg/ml透明质酸,离心;X组:加入1mlPBS,含4mg/ml透明质酸,离心;离心转速为1500rpm,10min,去上清,再加入100μlPBS重悬精子沉淀。
3、精子检测:
(1)试剂配制
FITC-PNA:称取1mg的FITC-PNA,(sigma公司,货号L7381),加入10mlPBS(Gibco公司,货号14190),将其配成0.1mg/ml浓储液,溶解好的浓储液分装后,使用锡箔纸包好储存在-20℃;
6-CFDA:称取4.6mg的6-CFDA,(sigma公司,货号C5014),加入10mlDMSO(sigma公司,货号D2650),将其配成0.46mg/ml浓储液,溶解好的浓储液分装后,使用锡箔纸包好储存在-20℃;
福尔马林配制:甲醛溶液与水按照3:7的比例混合;
(2)检测
①精子活力检测
预先把载玻片和盖玻片放在37℃加热板上预热10分钟,用移液器离心管取15μl上述步骤2精液稀释后的样品滴在载玻片上,压片后置于显微镜下镜检;
活力=活精子数/活精子数与死精子数的和
②精子顶体检测
<1取100μlPBS加入1.5ml离心管中,再加入0.6μl FITC-PNA,分别标上A、B、C、D、X;
<2在A组的管中加入200微升步骤2未离心的待测精子,B、C、D、X组各加入20微升步骤2离心后的待测精子;
<337℃避光染色20分,离心去上清,再加入一定量PBS悬浮精子;
<4取步骤<337℃染色的待测精子10μl滴片,加入2-3μl配制的福尔马林,盖片,荧光下观察,计数200个以上;
③精子膜完整性检测
<1取1mlPBS加入1.5ml离心管中,再加入20μl 6-CFDA,分别标上A、B、C、D、X;
<2在A组的管中加入200微升步骤2未离心的待测精子,B、C、D、X组各加入20微升步骤2离心后的待测精子;
<337℃避光染色20分,离心去上清,再加入一定量PBS悬浮精子;
<4取步骤<337℃染色的待测精子10μl滴片,加入2-3μl配制的福尔马林,盖片,荧光下观察。
实施例2:
1、透明质酸的配制
0.5mg/ml透明质酸溶液的配制:称取0.5mg透明质酸(国产)加入1ml的PBS中,37度5min,轻轻上下颠倒将液体混匀;
2mg/ml透明质酸溶液的配制:称取2mg透明质酸(国产)加入1ml的PBS中,37度5min,轻轻上下颠倒将液体混匀;
4mg/ml透明质酸溶液的配制:称取4mg透明质酸(国产)加入1ml的PBS中,37度5min,轻轻上下颠倒将液体混匀;
2、取4只冷冻羊精液,解冻后平均分为5份,每份100μl;A组:加入1mlPBS,不离心;B组:加入1mlPBS,离心;C组:加入1mlPBS,含0.5mg/ml透明质酸,离心;D组:加入1mlPBS,含2mg/ml透明质酸,离心;X组:加入1mlPBS,含4mg/ml透明质酸,离心;离心转速为2000rpm,10min,去上清,再加入100μlPBS重悬精子沉淀;
3、精子检测
(1)试剂配制
FITC-PNA:称取1mg的FITC-PNA,(sigma公司,货号L7381),加入10mlPBS(Gibco公司,货号14190),将其配成0.1mg/ml浓储液,溶解好的浓储液分装后,使用锡箔纸包好储存在-20℃;
6-CFDA:称取4.6mg的6-CFDA,(sigma公司,货号C5014),加入10mlDMSO(sigma公司,货号D2650),将其配成0.46mg/ml浓储液,溶解好的浓储液分装后,使用锡箔纸包好储存在-20℃;
福尔马林配制:甲醛溶液与水按照3:7的比例混合;
(2)检测
①精子活力检测
预先把载玻片和盖玻片放在37℃加热板上预热10分钟,用移液器离心管取15μl上述步骤2精液稀释后的样品滴在载玻片上,压片后置于显微镜下镜检;
活力=活精子数/活精子数与死精子数的和
②精子顶体检测
<1取100μlPBS加入1.5ml离心管中,再加入0.6μl FITC-PNA,分别标上A、B、C、D、X;
<2在A组的管中加入200微升步骤2未离心的待测精子,B、C、D、X组各加入20微升步骤2离心后的待测精子;
<337℃避光染色20分,离心去上清,再加入一定量PBS悬浮精子;
<4取步骤<337℃染色的待测精子10μl滴片,加入2-3μl配制的福尔马林,盖片,荧光下观察,计数200个以上。
③精子膜完整性检测
<1取1mlPBS加入1.5ml离心管中,再加入20μl 6-CFDA,分别标上A、B、C、D、X;
<2在A组的管中加入200微升步骤2未离心的待测精子,B、C、D、X组各加入20微升步骤2离心后的待测精子;
<337℃避光染色20分,离心去上清,再加入一定量PBS悬浮精子;
<4取步骤<337℃染色的待测精子10μl滴片,加入2-3μl配制的福尔马林,盖片,荧光下观察。
Claims (3)
1.一种透明质酸的用途,其特征在于:用于非治疗目的的降低精子离心损伤。
2.根据权利要求1所述透明质酸的用途,其特征在于:所述降低精子离心损伤是指降低离心对精子膜与顶体的损伤。
3.根据权利要求1所述透明质酸的用途,其特征在于:用于降低精子离心损伤的方法包括以下步骤:
(1)、透明质酸的配制
透明质酸溶液:每毫升精子洗涤液添加0.5mg~4mg的透明质酸,37度放置5min-10min,轻轻上下颠倒使透明质酸混匀;
(2)、将鲜精或冻精放入加入透明质酸的精子洗涤液中轻轻混匀后离心;
(3)、离心后吸去上清,将精子沉淀重悬。
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