CN115192521A - 一种褪黑素注射液的制备方法及其应用 - Google Patents
一种褪黑素注射液的制备方法及其应用 Download PDFInfo
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Abstract
本发明涉及生殖生物学技术领域，具体涉及一种褪黑素注射液的制备方法及其应用。使奶山羊在热应激的条件下，保护种公畜生殖热损伤，提高精液质量与受胎率，防控夏季高温对配种繁育的不利影响。本发明褪黑素注射液的制备方法为：1）取0.1—1g褪黑素粉末，置于1 mL乙醇溶液中，震荡使其全部溶解，制得褪黑素‑乙醇溶液；2）将1 mL褪黑素‑乙醇溶液与39 mL高压灭菌后的蒸馏水混匀，制得褪黑素‑乙醇水溶液并增加辅料制得褪黑素注射液。本发明使用褪黑素注射液有效缓解季节性高温引起的雄性奶山羊生殖损伤，可以有效地提高生产上奶山羊精液质量。为生产实践上使用外源褪黑素防控夏季高温对配种繁育的不利影响提供了参考。
Description
技术领域
本发明涉及生殖生物学技术领域，具体涉及一种褪黑素注射液的制备方法及其应用。
背景技术
夏季高温作为一种自然环境中的应激源，对畜牧生产造成的影响越来越大。对于动物体个体而言，阴囊和睾丸温度调节能力相对较差，种公畜在季节性高温下，不仅食欲减退、体重下降，还会使阴囊和睾丸温度升高。
热应激在临床表现上最直接的影响就是精子的受精能力和胚胎发育能力。体外受精实验发现，当精子受到热应激损伤后，精子顶体完整率下降，穿透透明带的速率和胚胎受精率都会显著降低，并且即使受精，胚胎发育迟缓占比也会升高，胚胎死亡率增加。
动物的睾丸温度始终比机体核心温度低2～4℃，这个温度对于精子发生非常重要。当机体发生热应激，引起体温升高，会导致阴囊温度升高，从而造成睾丸高热。精子发生需要适宜的内外环境，对温度尤为敏感，当温度相关因素发生改变，影响精子发生过程，就会对成熟精子产生不良影响。温度每上升1℃，精子发生率可能会下降14％，持续高热会导致睾丸萎缩和精子发生停滞甚至是睾丸变性，从而造成精子活力、密度下降，膜完整精子减少，直线前进型精子减少，顶体异常率升高，有效精子数减少等不良影响，最终导致受胎率下滑，生产效益下降。
发明内容
本发明提供一种褪黑素注射液的制备方法和应用。使奶山羊在热应激的条件下，保护种公畜生殖热损伤，提高精液质量与受胎率，防控夏季高温对配种繁育的不利影响。
与现有技术相比，本发明采用的技术方案为：一种褪黑素注射液的制备方法，其特征在于：步骤为：
1)取0.1—1g褪黑素粉末，置于1mL乙醇溶液中，震荡使其全部溶解，制得褪黑素-乙醇溶液；
2)将1mL褪黑素-乙醇溶液与39mL高压灭菌后的蒸馏水混匀，制得褪黑素-乙醇水溶液并增加辅料制得褪黑素注射液。
上述辅料的添加方法为：
1)制得褪黑素-乙醇水溶液后，加入0.03—0.06g抗氧化剂、0.003—0.007g金属离子络合剂、0.25—0.6g稳定剂，搅拌分散均匀；然后加入0.1—0.2mL抑菌剂、0.1—0.2mL肌内注射麻醉剂，搅拌分散均匀；
2)再加入0.001—0.002g缓冲剂，将溶液pH调节至6.0；
3)加入1.5—1.9g葡萄糖粉剂，将溶液渗透压调节至1.5％即可。
上述抗氧化剂为亚硫酸氢钠粉末。
4.如权利要求2或3所述的一种褪黑素注射液的制备方法，其特征在于，所述的金属离子络合剂为EDTA-Na2粉末。
上述稳定剂烟酰胺粉末。
上述抑菌剂苯酚溶液。
上述肌内注射麻醉剂为盐酸普鲁卡因溶液。
上述缓冲剂为碳酸氢钠粉末。
制备所得的褪黑素注射液在缓解奶山羊季节性生殖损伤中的应用。
与现有技术相比，本发明具有以下有益的技术效果：
1)本发明将褪黑素粉末制成注射液制剂，可以显著改善季节性热应激下雄性奶山羊的各项精液指标，有效缓解热应激造成的生殖损伤，提高精液质量。
2)本发明制得的褪黑素注射液有效地保护了季节性热应激下雄性奶山羊的生殖能力。褪黑素独特的安全性避免了许多不良反应。结果表明褪黑素注射液的使用，提高了高温下奶山羊精子的密度、活力、活率、前向性和有效精子数，减少了畸形率、顶体损伤率和膜损伤率。为褪黑素在保护雄性生殖的应用，防控了夏季高温对配种繁育的不利影响。
附图说明
图1为褪黑素制剂对奶山羊精子质量损伤的治疗结果；其中(A)精子密度指标水平；(B)精子活力指标水平；(C)精子活率指标水平；(D)精子前向性水平；(E)有效精子数占比；(F)精子畸形率水平；(G)精子膜结构完整率水平；(H)精子顶体完整率水平。
图2为褪黑素制剂对奶山羊睾丸损伤的治疗结果；其中(A)各组奶山羊睾丸组织切面HE染色结果；(B)各组分离、脱落和空泡化的曲细精管占比；(C)各组奶山羊睾丸组织切面凋亡染色结果；(D)各组凋亡细胞比率对比。
具体实施方式
下面结合具体的实施方式来对本发明的技术方案做进一步的限定：
实施例1：
一种褪黑素注射液的制备方法步骤为：
1)取0.3g褪黑素粉末，置于1mL乙醇溶液中，震荡使其全部溶解，制得褪黑素-乙醇溶液；
2)将1mL褪黑素-乙醇溶液与39mL高压灭菌后的蒸馏水混匀，制得褪黑素-乙醇水溶液并增加辅料制得褪黑素注射液。
上述辅料的添加方法为：
1)制得褪黑素-乙醇水溶液后，加入0.03g亚硫酸氢钠粉末、0.003gEDTA-Na2粉末、0.25g烟酰胺粉末，搅拌分散均匀；然后加入0.1mL苯酚溶液、0.1mL盐酸普鲁卡因溶液，搅拌分散均匀；
2)再加入0.001g碳酸氢钠粉末作为缓冲剂，将溶液pH调节至6.0；
3)加入1.5g葡萄糖粉剂，将溶液渗透压调节至1.5％即可。
实施例2：
一种褪黑素注射液的制备方法步骤为：
1)取0.2g褪黑素粉末，置于1mL乙醇溶液中，震荡使其全部溶解，制得褪黑素-乙醇溶液；
2)将1mL褪黑素-乙醇溶液与39mL高压灭菌后的蒸馏水混匀，制得褪黑素-乙醇水溶液并增加辅料制得褪黑素注射液。
上述辅料的添加方法为：
1)制得褪黑素-乙醇水溶液后，加入0.06g亚硫酸氢钠粉末、0.007gEDTA-Na2粉末、0.6g烟酰胺粉末，搅拌分散均匀；然后加入0.2mL苯酚溶液、0.2mL盐酸普鲁卡因溶液，搅拌分散均匀；
2)再加入0.002g碳酸氢钠粉末作为缓冲剂，将溶液pH调节至6.0；
3)加入1.9g葡萄糖粉剂，将溶液渗透压调节至1.5％即可。
实施例3：
一种褪黑素注射液的制备方法，其特征在于：步骤为：
1)取0.1g褪黑素粉末，置于1mL乙醇溶液中，震荡使其全部溶解，制得褪黑素-乙醇溶液；
2)将1mL褪黑素-乙醇溶液与39mL高压灭菌后的蒸馏水混匀，制得褪黑素-乙醇水溶液并增加辅料制得褪黑素注射液；
上述辅料的添加方法为：
1)制得褪黑素-乙醇水溶液后，加入0.04g亚硫酸氢钠粉末、0.004gEDTA-Na2粉末、0.5g烟酰胺粉末，搅拌分散均匀；然后加入0.2mL苯酚溶液、0.2mL盐酸普鲁卡因溶液，搅拌分散均匀；
2)然后加入0.002g碳酸氢钠粉末作为缓冲剂，将溶液PH调节至6.0；
3)加入1.8g葡萄糖粉剂，将溶液渗透压调节至1.5％。
本发明所制备的褪黑素注射液在缓解奶山羊季节性生殖损伤中应用。
褪黑素注射液使用浓度、剂量、注射方式和保存条件如下：
1)褪黑素分子终浓度为2.5mg/mL；
2)使用剂量为4mg/50kg体重；
3)使用方式为肌内注射；
4)制成褪黑素注射液，避光、4℃低温保存。
感官评价
取适量褪黑素注射液置于样品瓶中，观察其外观，对其分散性与浑浊度进行考察。对各项进行评分加和汇总，见表1。
表1评分标准
注：注射制剂性中，能够作为注射液为100分，不能作为注射液为0分；“——”表示没有内容。
实验结果
1、褪黑素注射剂改善精子质量的有效性评价
使用12只1岁6个月雄性萨能奶山羊，置于25*30m2的舍房中同条件饲养1周，自由进食与饮水。在夏季8月份进行为期十天的自然热应激造模，期间每天12:00-16:00将热应激组与治疗组奶山羊置于露天运动场自由活动。
动物分为自然对照组，热应激组与治疗组，共3组。造模期内治疗组奶山羊每日肌内注射4mg/50kg剂量实施例三褪黑素注射液，热应激组不处理。
使用人工阴道每三天收集一次精液(10次射精/月*2个月＝20次射精/只动物)。新鲜精液样本直接转移到实验室。共持续2个月，61天。使用迈朗动物精子分析仪评估以下指标：精子密度、精子活力、精子活率、精子前向性与有效精子数。使用光学显微镜进行以下形态学鉴定：精子畸形率、精子顶体完整率、精子膜完整率。形态学鉴定每份样本每项指标计数500个精子。
奶山羊精液质量热损伤变化观察：各组精液指标见图1。其中(A)为精子密度指标水平；(B)为精子活力指标水平；(C)为精子活率指标水平；(D)为精子前向性水平；(E)为有效精子数占比；(F)为精子畸形率水平；(G)为精子膜结构完整率水平；(H)为精子顶体完整率水平。
结果表明，热应激下奶山羊各项精液指标出现不同程度下降，精液质量持续低下。使用实施例3褪黑素注射液的实验组精液指标均有显著恢复，并且恢复节点早于热应激组。
2、褪黑素注射剂保护睾丸热损伤的有效性评价
造模处理后6天实验动物禁食，禁食后24h进行睾丸采样。肌肉注射30mg/kg剂量戊巴比妥钠麻醉，保定后消毒，剪开睾丸被膜，双端结扎割断睾丸系带，取下睾丸，切割适当大小组织块分别存放入4％PFA与液氮罐，用于后续形态学研究与基因和蛋白质表达分析。
各组睾丸切面情况见图2。其中，(A)为各组奶山羊睾丸组织切面HE染色结果；(B)为各组分离、脱落和空泡化的曲细精管占比；(C)为各组奶山羊睾丸组织切面凋亡染色结果；(D)为各组凋亡细胞比率对比。
结果表明，造模后7天奶山羊睾丸组织结构显著破坏，生精小管出现不同程度分离、脱落与空泡化，细胞出现显著凋亡反应，细胞层数减少、排列紊乱。使用实施例1褪黑素注射液的实验组睾丸组织结构稳定，凋亡反应显著下降，生精细胞层数增加，排列较为整齐。
Claims (9)
1.一种褪黑素注射液的制备方法，其特征在于：步骤为：
1)取0.1—1g褪黑素粉末，置于1mL乙醇溶液中，震荡使其全部溶解，制得褪黑素-乙醇溶液；
2)将1mL褪黑素-乙醇溶液与39mL高压灭菌后的蒸馏水混匀，制得褪黑素-乙醇水溶液并增加辅料制得褪黑素注射液。
2.如权利要求1所述的一种褪黑素注射液的制备方法，其特征在于，所述辅料的添加方法为：
1)制得褪黑素-乙醇水溶液后，加入0.03—0.06g抗氧化剂、0.003—0.007g金属离子络合剂、0.25—0.6g稳定剂，搅拌分散均匀；然后加入0.1—0.2mL抑菌剂、0.1—0.2mL肌内注射麻醉剂，搅拌分散均匀；
2)再加入0.001—0.002g缓冲剂，将溶液pH调节至6.0；
3)加入1.5—1.9g葡萄糖粉剂，将溶液渗透压调节至1.5％即可。
3.如权利要求2所述的一种褪黑素注射液的制备方法，其特征在于，所述的抗氧化剂为亚硫酸氢钠粉末。
4.如权利要求2或3所述的一种褪黑素注射液的制备方法，其特征在于，所述的金属离子络合剂为EDTA-Na2粉末。
5.如权利要求4所述的一种褪黑素注射液的制备方法，其特征在于，所述的稳定剂烟酰胺粉末。
6.如权利要求5所述的一种褪黑素注射液的制备方法，其特征在于，所述的抑菌剂苯酚溶液。
7.如权利要求6所述的一种褪黑素注射液的制备方法，其特征在于，所述的肌内注射麻醉剂为盐酸普鲁卡因溶液。
8.如权利要求7所述的一种褪黑素注射液的制备方法，其特征在于，所述的缓冲剂为碳酸氢钠粉末。
9.如权利要求1制备所得的褪黑素注射液在缓解奶山羊季节性生殖损伤中的应用。
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