三种渗透性抗冻剂和稀释基础液配合对兔精液冷冻保存效果的研究
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摘要
本试验以家兔为试验动物，从精液解冻后精子活率、顶体完整率、低渗膨胀阳
性率（HOST~＋）三个角度对三种渗透性抗冻剂（甘油、DMSO、乙酞胺）的抗冻效
果进行比较研究，并结合电镜下超微结构观察的结果进行分析。三种稀释基础液
（A：Tris3．52g，柠檬酸1．96g，葡萄糖1．59g，双蒸水100ml；B：Tris3．03g，柠
檬酸1.68g，葡萄糖1．25g，双蒸水1ooml;C：蔗糖5g，乳糖5g，双蒸水100ml）
与三种渗透性抗冻剂的三种浓度（4％，6％，8％）相配伍，共27组。将精液与稀
释液进行1：1稀释，4℃平衡3小时，－90－95℃冷冻10分钟，42℃20秒解冻。
结果表明（以下均指终浓度）：
1：不同的稀释基础液与不同的渗透性抗冻剂相配伍对冷冻解冻精子活率的影
响是不同的。在4％甘油＋A稀释基础液和5％乙酰胺＋C稀释基础液组解冻后精子
活率较高（P＜0.05）;在4％甘油＋c液和5％乙酰胺＋B液组解冻后精子活率较低
（P＜0.05）；DMSO对解冻精子活率的影响在本试验的浓度范围内与稀释基础液无
关。
2：不同的稀释基础液与不同的渗透性抗冻剂相配伍对解冻精液精子顶体完整
率的影响是不同的，在本试验基础液和浓度范围内乙酰胺的保护效果好一些。A、
B、C三种稀释基础液中乙酞胺组、DMSO组、甘油组的浓度间分别出现了保护效
果的不同（P＜0．05）。5％乙酰胺＋B稀释基础液对顶体的保护效果较好
（P＜0.05）；2％甘油十C稀释基础液对顶体的保护效果较差（P＜0．05）。
3：HOST~＋与精子活率正相关（r=O．7704）。低渗液组成：0．735g柠檬酸钠（三
水）、1．351g果糖，100毫升双蒸水。不同的稀释基础液与不同的渗透性抗冻剂相
配伍对解冻精液的精子膜功能完整性的影响是不同的，A液中三种抗冻剂对膜功能
完整性的保护效果差异不显著；B液中3％甘油优于4％乙酰胺（P＜o．05）；C液中
3％甘油优于3％DMSO（P＜0．05）。3％甘油与C液配伍对解冻精子膜功能完整性
的保护效果强于其它组，4％乙酰胺与B液配伍对解冻精子膜功能完整性的保护效
果较差（P＜0.05）。
4：冷冻复温后兔的一些精子不同程度出现了质膜膨胀、变薄、皱摺、及至破
坏；顶体肿胀、顶体外膜、内膜囊泡化或不连续，及至顶体完全完全脱落；部分精
子中段质膜破损，线粒体裸露、断裂、电子密度降低或部分丢失；少数主段末段质
膜破损，轴丝断裂、散开。
5 活率、顶体完整率、HOST三者的结果具有一致性。表明HOST可以作为检
验兔精液受精力的一个重要指标。
Glycerol, dimethyl suffixed, acetamide were adopted to verify the
cryoprotective ability of pentrating cryoprotectant with three kinds of
extenders.Sperm mobility, intact-acrosome ratio,plasma membrane int-
egrity(hypoosmotic swelling test,HOST) were assessed in fresh, cooled,
frozen-thawed sample and ultrastructural observation was carried out
to analyze the effect of frozed-thawed. Three extenders are as followed:
(1 )3.52%tris, 1.96%citric acid, 1.59%glucose;(2)3.03%tris, 1.68% citric
acid, 1.25%glucose; (3), 5%sucrose, 5%lactose, and three cryoprotect-
ants: glycerol and DMSO end concentration (2%,3%,4%) and acetamide
concentration (2%,4%, 5%). 1: ldiluention, equilibration at 4℃ for 3
hours,freezing at -90─95℃ for 10 minutes and thawing at 42℃ for 20
seconds .The results were as followed:
1:The influence on frozen-thawed sperm mobility is different when
differente extender is combined with different cryoprotectant.The perc-
entage of motile sperm is higher in 4% glycerol+A and 5%acetamide+
C(P<0.05);The mobility is lower in 4% glycerol +C and 5% acetamide
+ B(P<0.05).The influence of DMSO on frozen-thawed sperm mobility
has no relationship with diluents in this experiment(P>0.05).
2:The influence on intact-acrosome is different when different exte-
nder is combined with different cryoprotectant and acetamide had bett-
er protective effect than the others in designed concentrations(P<0.05).
With A,B and Cextender,different concentration of acetamide, DMSO
and glycerol had different effect on intact-acrosome ratio respectively
(P<0.05): The intact-acrosome ratio is higher in 5%acetamide + B. The
intact-acrosome ratio is lower in 2% glycerol+C (P<0.05).
3:The percentage of sperm tail swelling showed a significant
positive correlation with sperm motility (r=0.7704). With A extender,
three cryoprotectants had no significant different effect on frozen-
thawed sperm membrane functional integrity (P>0.05). With B diluent,
3%glycerol had better cryoprotective ability on membrane functional
integrity than 4%acetamide (P<0.05). With C diluent, 3% glycerol had
better cryoprotective effect on membrane functional integrity than
3%DMSO(P<0.05).HOS test can be used as a method to examine rabbit
sperm membrane functional integrity.
4:After freezing-thawing of semen, morphological changes of sperm
were that the plasma membrane became swelled, thined, folded,
disrupted and structural changes were that the acrosome swelled, the
outer or inner acrosomal membrane vesiculated and even lost. The
midpiece membrane of some sperm was disrupted, mitochondrion was
bared,cracked, and electric concentration is lower. The membrane of
principle and endpiece was disrupted and axoneme was cracked and
d is p ers ed.
5:Sperm mobility, intact-acrosome ratio, HOST positive ratio has
c o n s istency.
Master Candidate: Li Xiaoj uan
Specialty: Animal reproduction physiology
and animal reproduction technique
Supervisor: Professor Zhang Guixue
性率（HOST~＋）三个角度对三种渗透性抗冻剂（甘油、DMSO、乙酞胺）的抗冻效
果进行比较研究，并结合电镜下超微结构观察的结果进行分析。三种稀释基础液
（A：Tris3．52g，柠檬酸1．96g，葡萄糖1．59g，双蒸水100ml；B：Tris3．03g，柠
檬酸1.68g，葡萄糖1．25g，双蒸水1ooml;C：蔗糖5g，乳糖5g，双蒸水100ml）
与三种渗透性抗冻剂的三种浓度（4％，6％，8％）相配伍，共27组。将精液与稀
释液进行1：1稀释，4℃平衡3小时，－90－95℃冷冻10分钟，42℃20秒解冻。
结果表明（以下均指终浓度）：
1：不同的稀释基础液与不同的渗透性抗冻剂相配伍对冷冻解冻精子活率的影
响是不同的。在4％甘油＋A稀释基础液和5％乙酰胺＋C稀释基础液组解冻后精子
活率较高（P＜0.05）;在4％甘油＋c液和5％乙酰胺＋B液组解冻后精子活率较低
（P＜0.05）；DMSO对解冻精子活率的影响在本试验的浓度范围内与稀释基础液无
关。
2：不同的稀释基础液与不同的渗透性抗冻剂相配伍对解冻精液精子顶体完整
率的影响是不同的，在本试验基础液和浓度范围内乙酰胺的保护效果好一些。A、
B、C三种稀释基础液中乙酞胺组、DMSO组、甘油组的浓度间分别出现了保护效
果的不同（P＜0．05）。5％乙酰胺＋B稀释基础液对顶体的保护效果较好
（P＜0.05）；2％甘油十C稀释基础液对顶体的保护效果较差（P＜0．05）。
3：HOST~＋与精子活率正相关（r=O．7704）。低渗液组成：0．735g柠檬酸钠（三
水）、1．351g果糖，100毫升双蒸水。不同的稀释基础液与不同的渗透性抗冻剂相
配伍对解冻精液的精子膜功能完整性的影响是不同的，A液中三种抗冻剂对膜功能
完整性的保护效果差异不显著；B液中3％甘油优于4％乙酰胺（P＜o．05）；C液中
3％甘油优于3％DMSO（P＜0．05）。3％甘油与C液配伍对解冻精子膜功能完整性
的保护效果强于其它组，4％乙酰胺与B液配伍对解冻精子膜功能完整性的保护效
果较差（P＜0.05）。
4：冷冻复温后兔的一些精子不同程度出现了质膜膨胀、变薄、皱摺、及至破
坏；顶体肿胀、顶体外膜、内膜囊泡化或不连续，及至顶体完全完全脱落；部分精
子中段质膜破损，线粒体裸露、断裂、电子密度降低或部分丢失；少数主段末段质
膜破损，轴丝断裂、散开。
5 活率、顶体完整率、HOST三者的结果具有一致性。表明HOST可以作为检
验兔精液受精力的一个重要指标。
Glycerol, dimethyl suffixed, acetamide were adopted to verify the
cryoprotective ability of pentrating cryoprotectant with three kinds of
extenders.Sperm mobility, intact-acrosome ratio,plasma membrane int-
egrity(hypoosmotic swelling test,HOST) were assessed in fresh, cooled,
frozen-thawed sample and ultrastructural observation was carried out
to analyze the effect of frozed-thawed. Three extenders are as followed:
(1 )3.52%tris, 1.96%citric acid, 1.59%glucose;(2)3.03%tris, 1.68% citric
acid, 1.25%glucose; (3), 5%sucrose, 5%lactose, and three cryoprotect-
ants: glycerol and DMSO end concentration (2%,3%,4%) and acetamide
concentration (2%,4%, 5%). 1: ldiluention, equilibration at 4℃ for 3
hours,freezing at -90─95℃ for 10 minutes and thawing at 42℃ for 20
seconds .The results were as followed:
1:The influence on frozen-thawed sperm mobility is different when
differente extender is combined with different cryoprotectant.The perc-
entage of motile sperm is higher in 4% glycerol+A and 5%acetamide+
C(P<0.05);The mobility is lower in 4% glycerol +C and 5% acetamide
+ B(P<0.05).The influence of DMSO on frozen-thawed sperm mobility
has no relationship with diluents in this experiment(P>0.05).
2:The influence on intact-acrosome is different when different exte-
nder is combined with different cryoprotectant and acetamide had bett-
er protective effect than the others in designed concentrations(P<0.05).
With A,B and Cextender,different concentration of acetamide, DMSO
and glycerol had different effect on intact-acrosome ratio respectively
(P<0.05): The intact-acrosome ratio is higher in 5%acetamide + B. The
intact-acrosome ratio is lower in 2% glycerol+C (P<0.05).
3:The percentage of sperm tail swelling showed a significant
positive correlation with sperm motility (r=0.7704). With A extender,
three cryoprotectants had no significant different effect on frozen-
thawed sperm membrane functional integrity (P>0.05). With B diluent,
3%glycerol had better cryoprotective ability on membrane functional
integrity than 4%acetamide (P<0.05). With C diluent, 3% glycerol had
better cryoprotective effect on membrane functional integrity than
3%DMSO(P<0.05).HOS test can be used as a method to examine rabbit
sperm membrane functional integrity.
4:After freezing-thawing of semen, morphological changes of sperm
were that the plasma membrane became swelled, thined, folded,
disrupted and structural changes were that the acrosome swelled, the
outer or inner acrosomal membrane vesiculated and even lost. The
midpiece membrane of some sperm was disrupted, mitochondrion was
bared,cracked, and electric concentration is lower. The membrane of
principle and endpiece was disrupted and axoneme was cracked and
d is p ers ed.
5:Sperm mobility, intact-acrosome ratio, HOST positive ratio has
c o n s istency.
Master Candidate: Li Xiaoj uan
Specialty: Animal reproduction physiology
and animal reproduction technique
Supervisor: Professor Zhang Guixue
引文
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