猪精液冷冻保存技术研究
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摘要
采用手握法采集成年大约克种公猪精液,收集中段富含精子部分,配制不同的稀释液,在不同条件
下进行冻前预处理,再用不同的解冻液和解冻方法进行解冻,然后进行人工授精试验;同时,还对冷冻
前后精液中细菌含量与种类和低温保存猪精液进行了研究。结果如下:
1.在使用的 7 种稀释液中,5、6 号稀释液优于其它 5 种(P<0.05),其冻后活率分别达 0.46,0.50
(0.5ml 颗粒)和 0.40,0.49(0.5ml 细管),精子复苏率分别是 76.7%,74.6%(0.5ml 颗粒)和 70.5%,
78.3%(0.5ml 细管),且两者差异不显著(P>0.05);同种稀释液之间,颗粒与细管冻精的差异不显著
(P>0.05)。以颗粒冻精按不同的始冻温度、平衡温度、入氮温度制定了始冻温度与冻后活率的关系曲线
和冷冻温度曲线。
2.对新鲜精液在离心前静置不同时间,选择不同的离心速度,离心时间及不同的精清留量等进行冷
冻前预处理。结果表明:离心前在 37℃环境下静置 60min,其冻后效果(0.5ml 细管精液冻后活率达 0.50,
复苏率 75.8%,颗粒精液 0.5ml 冻后活率 0.48,复苏率 72.5%,0.1ml 活率 0.47,复苏率 70.6%)优于30min
和 90min(P<0.05);离心过程以 1000r/min 的速度离心 10min 较好(0.5ml 细管精液冻后活率 0.48,复
苏率 76.8%,0.5ml 颗粒精液冻后活率 0.46,复苏率 73.5%,0.1ml 颗粒冻后活率 0.44,复苏率 69.4%),
优于 500r/min 和 1500r/min 分别离心 7min 和 13min(P <0.05);精清留量以精清/精液(体积比)等于 1/1
时较好,优于 1/2 和 0/1(P <0.05)。
3.对五种不同的冷冻保护剂(甘油、DMSO、乙二醇、Tris、OEP)和五种自行设计的解冻液配方以及
解冻方法进行对比。结果表明:甘油为最佳的冷冻保护剂(P<0.05),其最适宜浓度为 3%(P<0.05),
冻后活率最高可达 0.48;不同冷冻保护剂对精子的毒性大小依次为:乙二醇>DMSO>OEP>Tris>甘油;4
号解冻液和精清的解冻效果明显好于其它三种解冻液(P<0.05),而两者之间无明显差异(P>.05),但
以 4 号解冻液在冻后活率方面稍高于精清;解冻方法以在 75℃下,4s 解冻效果较好(活率 0.47,顶体
完整率 55.6%,精子畸形率 18.32%)。
4.采用自制冷冻稀释液和解冻液配方,对自制的颗粒冻精和细管冻精进行人工授精试验。结果表明:
在有效存活时间和冻后活率上,颗粒冻精与细管冻精之间差异不明显(P>0.05);而颗粒冻精的受胎率和
窝均产仔数要好于细管冻精,分别为颗粒冻精:81.81%,85.71%,9.22 和 9.33 头,细管冻精 75.00%,
80.00%,8.17 和 8.25 头。
5.原精中细菌菌落数量平均为 1.5×104个/ml,平衡后精液中细菌菌落数量平均为 1.1×104个/ml;
颗粒冷冻精液中:0.1ml 含细菌菌落数 920 个,0.5ml 中有 3750 个;细管冷冻精液中:0.25ml 细管含细
菌菌落数量 1060 个,0.5ml 细管含细菌菌落数 1520 个。细菌经培养、染色后镜检,从外形上初步确定有
葡萄球菌、大肠杆菌和链球菌等。
6.用 5 种不同的低温保存稀释液按不同比例稀释,在 0℃～5℃的低温环境下保存。结果表明:2 号
稀释液的有效保存时间达 60±4.05h,与其它四种稀释液比较,差异极显著(P<0.01),猪精液低温保存离
心速度以 1000r/min 的离心效果较好,有效保存时间达 50±3.68h(P<0.05),同种稀释液不同比例稀释,
精液与稀释液按 1:1 的比例稀释,效果较好(P<0.05)。
Using hand-holding method to collect the York boar semen in this test,gather the middle
part of abundent sperm,confecting different diluents , pre-treating it with different
conditions , thawing with different liquids and ways, after that to inseminate;at the same
time , there are also investigating the microbe contents and sorts and the learning
low-temperature preservation of boar semen. The results were following:
1.In seven diluents,the liquids of No.5 and No.6 were better than others (P<0.05),the
after freezing mobility were 0.46、0.50(pellet) and 0.40、0.49(tubule), recovery percent
were 76.7%、74.6%(pellet) and 70.5%、78.3%(tubule), the difference between No.5 and No.6
was not notability(P>0.05);in same diluents ,there were no difference between pellet
freezing-sperm and tubule freezing-sperm(P>0.05)。The ideal frozen temperature curve and
the relation curve of beginning temperature and after-freezing mobility in pellet
freezing-sperm were set down with different beginning frozen temperature 、 balance
temperature 、and enter nitrogen temperature.
2.The pretreatment before freezing of setting different time、different ratio and time
of centrifugal、different remnant of liquid on boar novelty semen were made, compared the
effect of sperm freezing result on the different pretreatment. The result is following:
the result before centrifuging of setting 60min in 37℃ (0.5ml tubule after-sperm motility
0.50 ,recovery percent 75.8%, 0.5ml pellet after-sperm motility 0.48 , recovery percent 72.5%,
0.1ml pellet after sperm motility 0.47,recovery percent 70.6%)was better than 30min and 90min
(P<0.05);the centrifuging ratio of 1000r/min for 10min (0.5ml tubule after-sperm motility
0.48 ,recovery percent 76.8%, 0.5ml pellet after-sperm motility 0.46 , recovery percent 73.5%,
0.1ml pellet after sperm motility 0.44,recovery percent 69.4%)is better than 500r/min and
1500r/min for 7min and 13min(P<0.05);it is the best that the remnant of liquid is 1/1 than
1/2 and 0/1(P<0.05)。
3.Compared with different cryoprotectant (Glycerol、DMSO、Glycol、Tris、OEP) and different
thaw-liquids design by ourselves and thawing methods. The results showed:Glycerol is the
best cryoprotectant (P<0.05) and the best content is 3% (sperm motility 0.48)(p<0.05);
the sequence of toxicity of different cryoprotectant is :Glycol> DMSO> OEP> Tris> Glycerol;
the effect of the No.4 and the sperm liquids is better than others in evidence(P
but there is no distinctive diversity(P>.05);it is the better of thawing methods with
75℃,4s(motility 0.47,acorosomal integrity 55.6%,sperm abnormality 18.32%)。
4. Using pellet and tubule sperm to inseminate test that freezing diluents and thaw liquids
made by ourselves. The result showed :pellet and tubule sperm hadn’t distinctive difference
in effective living time and after thawn sperm(P>0.05);but the pellet sperm was better than
tubule in percent fecundation and equal litters , the pellet sperm was 81.81%,85.71%,9.22
and 9.33,tubule sperm was75.00%,80.00%,8.17 and 8.25。
5.The average number of microbe in pre-semen and after balance were 1.5×104 /ml、1.1
×104/ml;the average number of microbe in pellet freezing sperm (0.1ml、0.5ml) were 920 and
3750 , in tubule freezing sperm the number (0.25ml、0.5ml) were 1060 and 1520.The sorts of
microbe had Grape、Intestines bacillus、Streptococcus and so on.
6.It was persevered by different extender with different ratio of low temperature
preservation in 0℃～5℃.The result showed: the No. 2 could persevere 60±4.05h effective ,
there was very nobility difference with the others (P<0.01);The centrifuging ratio was better
with 1000r/min and could persevere 50±3.68h effective (P<0.05)in low temperature. The ratio
of diluents was better with 1:1(semen/diluents).
下进行冻前预处理,再用不同的解冻液和解冻方法进行解冻,然后进行人工授精试验;同时,还对冷冻
前后精液中细菌含量与种类和低温保存猪精液进行了研究。结果如下:
1.在使用的 7 种稀释液中,5、6 号稀释液优于其它 5 种(P<0.05),其冻后活率分别达 0.46,0.50
(0.5ml 颗粒)和 0.40,0.49(0.5ml 细管),精子复苏率分别是 76.7%,74.6%(0.5ml 颗粒)和 70.5%,
78.3%(0.5ml 细管),且两者差异不显著(P>0.05);同种稀释液之间,颗粒与细管冻精的差异不显著
(P>0.05)。以颗粒冻精按不同的始冻温度、平衡温度、入氮温度制定了始冻温度与冻后活率的关系曲线
和冷冻温度曲线。
2.对新鲜精液在离心前静置不同时间,选择不同的离心速度,离心时间及不同的精清留量等进行冷
冻前预处理。结果表明:离心前在 37℃环境下静置 60min,其冻后效果(0.5ml 细管精液冻后活率达 0.50,
复苏率 75.8%,颗粒精液 0.5ml 冻后活率 0.48,复苏率 72.5%,0.1ml 活率 0.47,复苏率 70.6%)优于30min
和 90min(P<0.05);离心过程以 1000r/min 的速度离心 10min 较好(0.5ml 细管精液冻后活率 0.48,复
苏率 76.8%,0.5ml 颗粒精液冻后活率 0.46,复苏率 73.5%,0.1ml 颗粒冻后活率 0.44,复苏率 69.4%),
优于 500r/min 和 1500r/min 分别离心 7min 和 13min(P <0.05);精清留量以精清/精液(体积比)等于 1/1
时较好,优于 1/2 和 0/1(P <0.05)。
3.对五种不同的冷冻保护剂(甘油、DMSO、乙二醇、Tris、OEP)和五种自行设计的解冻液配方以及
解冻方法进行对比。结果表明:甘油为最佳的冷冻保护剂(P<0.05),其最适宜浓度为 3%(P<0.05),
冻后活率最高可达 0.48;不同冷冻保护剂对精子的毒性大小依次为:乙二醇>DMSO>OEP>Tris>甘油;4
号解冻液和精清的解冻效果明显好于其它三种解冻液(P<0.05),而两者之间无明显差异(P>.05),但
以 4 号解冻液在冻后活率方面稍高于精清;解冻方法以在 75℃下,4s 解冻效果较好(活率 0.47,顶体
完整率 55.6%,精子畸形率 18.32%)。
4.采用自制冷冻稀释液和解冻液配方,对自制的颗粒冻精和细管冻精进行人工授精试验。结果表明:
在有效存活时间和冻后活率上,颗粒冻精与细管冻精之间差异不明显(P>0.05);而颗粒冻精的受胎率和
窝均产仔数要好于细管冻精,分别为颗粒冻精:81.81%,85.71%,9.22 和 9.33 头,细管冻精 75.00%,
80.00%,8.17 和 8.25 头。
5.原精中细菌菌落数量平均为 1.5×104个/ml,平衡后精液中细菌菌落数量平均为 1.1×104个/ml;
颗粒冷冻精液中:0.1ml 含细菌菌落数 920 个,0.5ml 中有 3750 个;细管冷冻精液中:0.25ml 细管含细
菌菌落数量 1060 个,0.5ml 细管含细菌菌落数 1520 个。细菌经培养、染色后镜检,从外形上初步确定有
葡萄球菌、大肠杆菌和链球菌等。
6.用 5 种不同的低温保存稀释液按不同比例稀释,在 0℃～5℃的低温环境下保存。结果表明:2 号
稀释液的有效保存时间达 60±4.05h,与其它四种稀释液比较,差异极显著(P<0.01),猪精液低温保存离
心速度以 1000r/min 的离心效果较好,有效保存时间达 50±3.68h(P<0.05),同种稀释液不同比例稀释,
精液与稀释液按 1:1 的比例稀释,效果较好(P<0.05)。
Using hand-holding method to collect the York boar semen in this test,gather the middle
part of abundent sperm,confecting different diluents , pre-treating it with different
conditions , thawing with different liquids and ways, after that to inseminate;at the same
time , there are also investigating the microbe contents and sorts and the learning
low-temperature preservation of boar semen. The results were following:
1.In seven diluents,the liquids of No.5 and No.6 were better than others (P<0.05),the
after freezing mobility were 0.46、0.50(pellet) and 0.40、0.49(tubule), recovery percent
were 76.7%、74.6%(pellet) and 70.5%、78.3%(tubule), the difference between No.5 and No.6
was not notability(P>0.05);in same diluents ,there were no difference between pellet
freezing-sperm and tubule freezing-sperm(P>0.05)。The ideal frozen temperature curve and
the relation curve of beginning temperature and after-freezing mobility in pellet
freezing-sperm were set down with different beginning frozen temperature 、 balance
temperature 、and enter nitrogen temperature.
2.The pretreatment before freezing of setting different time、different ratio and time
of centrifugal、different remnant of liquid on boar novelty semen were made, compared the
effect of sperm freezing result on the different pretreatment. The result is following:
the result before centrifuging of setting 60min in 37℃ (0.5ml tubule after-sperm motility
0.50 ,recovery percent 75.8%, 0.5ml pellet after-sperm motility 0.48 , recovery percent 72.5%,
0.1ml pellet after sperm motility 0.47,recovery percent 70.6%)was better than 30min and 90min
(P<0.05);the centrifuging ratio of 1000r/min for 10min (0.5ml tubule after-sperm motility
0.48 ,recovery percent 76.8%, 0.5ml pellet after-sperm motility 0.46 , recovery percent 73.5%,
0.1ml pellet after sperm motility 0.44,recovery percent 69.4%)is better than 500r/min and
1500r/min for 7min and 13min(P<0.05);it is the best that the remnant of liquid is 1/1 than
1/2 and 0/1(P<0.05)。
3.Compared with different cryoprotectant (Glycerol、DMSO、Glycol、Tris、OEP) and different
thaw-liquids design by ourselves and thawing methods. The results showed:Glycerol is the
best cryoprotectant (P<0.05) and the best content is 3% (sperm motility 0.48)(p<0.05);
the sequence of toxicity of different cryoprotectant is :Glycol> DMSO> OEP> Tris> Glycerol;
the effect of the No.4 and the sperm liquids is better than others in evidence(P
but there is no distinctive diversity(P>.05);it is the better of thawing methods with
75℃,4s(motility 0.47,acorosomal integrity 55.6%,sperm abnormality 18.32%)。
4. Using pellet and tubule sperm to inseminate test that freezing diluents and thaw liquids
made by ourselves. The result showed :pellet and tubule sperm hadn’t distinctive difference
in effective living time and after thawn sperm(P>0.05);but the pellet sperm was better than
tubule in percent fecundation and equal litters , the pellet sperm was 81.81%,85.71%,9.22
and 9.33,tubule sperm was75.00%,80.00%,8.17 and 8.25。
5.The average number of microbe in pre-semen and after balance were 1.5×104 /ml、1.1
×104/ml;the average number of microbe in pellet freezing sperm (0.1ml、0.5ml) were 920 and
3750 , in tubule freezing sperm the number (0.25ml、0.5ml) were 1060 and 1520.The sorts of
microbe had Grape、Intestines bacillus、Streptococcus and so on.
6.It was persevered by different extender with different ratio of low temperature
preservation in 0℃～5℃.The result showed: the No. 2 could persevere 60±4.05h effective ,
there was very nobility difference with the others (P<0.01);The centrifuging ratio was better
with 1000r/min and could persevere 50±3.68h effective (P<0.05)in low temperature. The ratio
of diluents was better with 1:1(semen/diluents).
引文
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