不同浓度卵黄离心及精液透析处理对猪精子冷冻效果的影响
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摘要
为了提高猪冷冻精液解冻后的品质,手握法采集成年种公猪精液,采用不同的方式对冷冻液进行预处理,冷冻,解冻后检测猪精液的精子品质,并在此基础上做体外受精试验,观察胚胎的发育效果,以为猪的高效繁殖生产提供一定理论基础。本论文共分为4个试验,其试验方法和结果如下:
试验一卵黄离心处理对猪精液冷冻效果的影响
研究冷冻Ⅰ液不同卵黄比例离心处理后对猪精液品质的影响,卵黄在冷冻Ⅰ液中的浓度分别为20%、30%、40%和50%。解冻后通过CASA精子分析系统来评定活动精子百分率、活率和精子直线运动速度,以及采用HOST法与FITC-PNA染色法分别来评定精子质膜完整率和顶体完整率。结果分析表明:在配制冷冻Ⅰ液前对卵黄进行离心处理有助于改善冷冻稀释液保护冷冻猪精子效果,与对照组相比较,除50%离心卵黄组外,各试验组的精子运动参数、质膜完整率及顶体完整率均优于对照组(P<0.05)。20%离心卵黄组和30%离心卵黄组的精子运动参数显著优于对照组(P<0.05);30%离心卵黄组的质膜完整率和顶体完整率均显著高于对照组(P<0.05)。综合本试验所测定的各项冷冻猪精子的评价指标,30%离心卵黄组的冷冻猪精子在本试验条件下品质最佳。
试验二精液透析处理对猪精液冷冻效果的影响
研究在加入冷冻Ⅰ液前进行精液透析处理对猪精液品质的影响,透析袋的规格为12-14 KD。解冻后评定活动精子百分率、活率和精子直线运动速度;精子质膜完整率和顶体完整率。结果分析表明:精液透析处理组的活动精子百分率、精子活率、质膜完整率和顶体完整率均明显优于对照组,尤其冻后精子活率改善最为明显(P<0.05),较对照组提高了21.75%。说明使用透析袋对猪精液进行降温平衡处理对冷冻猪精子有良好的保护效果。
试验三添加抗氧化剂对猪精液冷冻效果的影响
研究在冷冻稀释Ⅰ液中添加VC 10 mmol/ml、VE 0.2 mg/ml和联合添加10 mmol/ml Vc和0.2 mg/ml VE对猪精液品质的影响,解冻后评定活动精子百分率、活率和精子直线运动速度;精子质膜完整率和顶体完整率。结果表明:在冷冻稀释Ⅰ液中添加VE 0.2 mg/ml和联合添加10 mmol/ml Vc和0.2 mg/ml VE对猪冷冻精子解冻后的精子品质有一定的改善作用,联合添加组与单独添加VE组没有显著差异。
试验四猪冷冻精液的体外受精试验
在试验一、二、三的基础上进行猪精液的体外受精试验,以新鲜精液作对照,观察胚胎的发育情况。评价指标为卵裂细胞数,2-细胞率、8-细胞率、桑椹胚发育率和囊胚发育率。结果分析表明:冷冻精液和新鲜精液无论从2-细胞率、8-细胞、桑椹胚发育率还是囊胚发育率上均无显著差异( P >0.05)。表明本研究的体外受精方法适合用于冻精的体外受精。
In order to improve the quality of boar frozen-thawed semen, hand-holding method was used to collect boar semen, using different method to pretreat with the o freezing liquid, and detecting the quality of semen spermatozoa after thawing.We did the in vitro fertilization on the basis of the experiment results and observe the development of the embryonic. The purposes of the thesis were to provide some basic theory to improve the efficient of pig breeding and production. This paper was divided into three tests, the method and the results were summarized as follows:
Experiment 1. The effects of centrifugation method for egg yolk on freezing of boar semen
Research the effects of different concentration of egg yolk centrifugation inⅠfreezing liquid on the quality of the boar frozen-thawed semen.The concentration of egg yolk were 20%, 30%, 40% and 50%. We evaluated the TM, Motility and VSL of sperm motion through the CASA analyzing system; and assessed the Plasma Integrity, Normal Acrosome by FITC and PNA - HOST staining method respectively. Results indicated that: the egg yolk centrifugation method before madeup theⅠfreezing liquid could improve the protection effectiveness of diluted-frozen extenders on boar semen. The sperm motion, Plasma Integrity and Normal Acrosome of the treatment groups were all significant better than the control group, except the 50% centrifugal yolk treatment (P<0.05); The sperm motion of the 20% centrifugal yolk treatment and the 30% centrifugal yolk treatment were higher than the control group (P<0.05); and the Plasma Integrity and Normal Acrosome of the 30% centrifugal yolk treatment were significant higher than the control group (P<0.05). According to the comprehensive evaluation index of the frozen semen, the quailty of the 30% centrifugal yolk treatment was the best at the experiment condition.
Experiment 2. The effects of dialysis of semen on freezing of boar semen
Research the effects of semen dialysis on the quailty of boar frozen-thawed semen, the speecification of the dialysis bag is 12-14 KD. We evaluated the TM, Motility, VSL, the Plasma Integrity and Normal Acrosome of the boar spermatozoa. The results showed: The dialysis treatment could significant improve the TM, Motility, the Plasma Integrity and Normal Acrosome,compared to the control group,especially the TM, which was increased by 21.75%. So, the method of dialysis had good protection effect to the boar frozen-thawed semen.
Experiment 3. The effects of antioxidants supplementation on freezing of boar semen
Research the effects of 10 mmol/ml VC, 0.2 mg/m VE and both 10 mmol/ml VC and 0.2 mg/m VE supplemented inⅠfreezing liquid on freezing of boar semen. We evaluated the TM, Motility, VSL, the Plasma Integrity and Normal Acrosome of the boar spermatozoa. The results showed: supplementation of VE 0.2 mg/ml, both 10 mmol/ml VC and 0.2 mg/ml VE had some improvement on the quailty of boar frozen-thawed semen, but the simltaneity supplementation group had no significant difference comparing to the VE group.
Experiment 4. The IVF experiment using the frozen-thawed boar semen.
Using the fresh-ejaculated spermatozoa as the control group, we did the IVF on the basis of the experiment 1、2 and 3: the 30% centrifugal yolk treatment and the dialysis treatment,and observed the development of the embryo. The number of treatmented oocytes, 2-cell rate, 8-cell rate, Morula rate, Bastocysts rate was investigated. The results showed no significant difference with fresh-djaculated semen at all the measurement indicators. Then, the pre-treated method was suitable for the IVF of the boar frozen-thawed semen.
试验一卵黄离心处理对猪精液冷冻效果的影响
研究冷冻Ⅰ液不同卵黄比例离心处理后对猪精液品质的影响,卵黄在冷冻Ⅰ液中的浓度分别为20%、30%、40%和50%。解冻后通过CASA精子分析系统来评定活动精子百分率、活率和精子直线运动速度,以及采用HOST法与FITC-PNA染色法分别来评定精子质膜完整率和顶体完整率。结果分析表明:在配制冷冻Ⅰ液前对卵黄进行离心处理有助于改善冷冻稀释液保护冷冻猪精子效果,与对照组相比较,除50%离心卵黄组外,各试验组的精子运动参数、质膜完整率及顶体完整率均优于对照组(P<0.05)。20%离心卵黄组和30%离心卵黄组的精子运动参数显著优于对照组(P<0.05);30%离心卵黄组的质膜完整率和顶体完整率均显著高于对照组(P<0.05)。综合本试验所测定的各项冷冻猪精子的评价指标,30%离心卵黄组的冷冻猪精子在本试验条件下品质最佳。
试验二精液透析处理对猪精液冷冻效果的影响
研究在加入冷冻Ⅰ液前进行精液透析处理对猪精液品质的影响,透析袋的规格为12-14 KD。解冻后评定活动精子百分率、活率和精子直线运动速度;精子质膜完整率和顶体完整率。结果分析表明:精液透析处理组的活动精子百分率、精子活率、质膜完整率和顶体完整率均明显优于对照组,尤其冻后精子活率改善最为明显(P<0.05),较对照组提高了21.75%。说明使用透析袋对猪精液进行降温平衡处理对冷冻猪精子有良好的保护效果。
试验三添加抗氧化剂对猪精液冷冻效果的影响
研究在冷冻稀释Ⅰ液中添加VC 10 mmol/ml、VE 0.2 mg/ml和联合添加10 mmol/ml Vc和0.2 mg/ml VE对猪精液品质的影响,解冻后评定活动精子百分率、活率和精子直线运动速度;精子质膜完整率和顶体完整率。结果表明:在冷冻稀释Ⅰ液中添加VE 0.2 mg/ml和联合添加10 mmol/ml Vc和0.2 mg/ml VE对猪冷冻精子解冻后的精子品质有一定的改善作用,联合添加组与单独添加VE组没有显著差异。
试验四猪冷冻精液的体外受精试验
在试验一、二、三的基础上进行猪精液的体外受精试验,以新鲜精液作对照,观察胚胎的发育情况。评价指标为卵裂细胞数,2-细胞率、8-细胞率、桑椹胚发育率和囊胚发育率。结果分析表明:冷冻精液和新鲜精液无论从2-细胞率、8-细胞、桑椹胚发育率还是囊胚发育率上均无显著差异( P >0.05)。表明本研究的体外受精方法适合用于冻精的体外受精。
In order to improve the quality of boar frozen-thawed semen, hand-holding method was used to collect boar semen, using different method to pretreat with the o freezing liquid, and detecting the quality of semen spermatozoa after thawing.We did the in vitro fertilization on the basis of the experiment results and observe the development of the embryonic. The purposes of the thesis were to provide some basic theory to improve the efficient of pig breeding and production. This paper was divided into three tests, the method and the results were summarized as follows:
Experiment 1. The effects of centrifugation method for egg yolk on freezing of boar semen
Research the effects of different concentration of egg yolk centrifugation inⅠfreezing liquid on the quality of the boar frozen-thawed semen.The concentration of egg yolk were 20%, 30%, 40% and 50%. We evaluated the TM, Motility and VSL of sperm motion through the CASA analyzing system; and assessed the Plasma Integrity, Normal Acrosome by FITC and PNA - HOST staining method respectively. Results indicated that: the egg yolk centrifugation method before madeup theⅠfreezing liquid could improve the protection effectiveness of diluted-frozen extenders on boar semen. The sperm motion, Plasma Integrity and Normal Acrosome of the treatment groups were all significant better than the control group, except the 50% centrifugal yolk treatment (P<0.05); The sperm motion of the 20% centrifugal yolk treatment and the 30% centrifugal yolk treatment were higher than the control group (P<0.05); and the Plasma Integrity and Normal Acrosome of the 30% centrifugal yolk treatment were significant higher than the control group (P<0.05). According to the comprehensive evaluation index of the frozen semen, the quailty of the 30% centrifugal yolk treatment was the best at the experiment condition.
Experiment 2. The effects of dialysis of semen on freezing of boar semen
Research the effects of semen dialysis on the quailty of boar frozen-thawed semen, the speecification of the dialysis bag is 12-14 KD. We evaluated the TM, Motility, VSL, the Plasma Integrity and Normal Acrosome of the boar spermatozoa. The results showed: The dialysis treatment could significant improve the TM, Motility, the Plasma Integrity and Normal Acrosome,compared to the control group,especially the TM, which was increased by 21.75%. So, the method of dialysis had good protection effect to the boar frozen-thawed semen.
Experiment 3. The effects of antioxidants supplementation on freezing of boar semen
Research the effects of 10 mmol/ml VC, 0.2 mg/m VE and both 10 mmol/ml VC and 0.2 mg/m VE supplemented inⅠfreezing liquid on freezing of boar semen. We evaluated the TM, Motility, VSL, the Plasma Integrity and Normal Acrosome of the boar spermatozoa. The results showed: supplementation of VE 0.2 mg/ml, both 10 mmol/ml VC and 0.2 mg/ml VE had some improvement on the quailty of boar frozen-thawed semen, but the simltaneity supplementation group had no significant difference comparing to the VE group.
Experiment 4. The IVF experiment using the frozen-thawed boar semen.
Using the fresh-ejaculated spermatozoa as the control group, we did the IVF on the basis of the experiment 1、2 and 3: the 30% centrifugal yolk treatment and the dialysis treatment,and observed the development of the embryo. The number of treatmented oocytes, 2-cell rate, 8-cell rate, Morula rate, Bastocysts rate was investigated. The results showed no significant difference with fresh-djaculated semen at all the measurement indicators. Then, the pre-treated method was suitable for the IVF of the boar frozen-thawed semen.
引文
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