猪精液冷冻保存技术研究
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摘要
猪精液冷冻技术研究是随着猪人工授精技术的广泛应用而发展起来的,被认为是养猪业发展的一个重要里程碑。精液冷冻技术使建立一个不受时间、地域限制的“精子库”成为可能,可为大量母猪提供携带优秀遗传资源的精子。此外,猪冷冻精液还可为猪的育种提供生物安全保障。冷冻保存精子的受精能力是影响母畜保持高繁殖力的重要因素。但由于猪精子具有一定的生理特殊性,其冷冻保存效果与牛相比还有相当大的差距,目前仍未形成一套有效的保存技术体系,在今后相当的时间内仍不能应用到养猪业生产中。
为了进一步改善猪冷冻精子的品质,本研究在改进猪精液冷冻方法与优化冷冻稀释液两个方面进行了研究,以精子活率、线粒体活性、精子质膜完整性、精子项体完整率和低渗膨胀测试(HOST)等为指标,利用精子自动分析仪(CASA)结合荧光染色方法及一些常规检测手段对精子品质进行了客观有效的评价。
本论文主要针对以下五个方面进行研究:
试验一,N,N-二甲基甲酰胺(DMF)提高猪精液冷冻效果研究。近几年来,一些研究者将DMF用于家畜精子冷冻中,在一些家畜精液冷冻中进行了尝试,并取得优于甘油的效果。然而,DMF在猪精液冷冻中的研究却很少报道。本试验以DMF和甘油组成混合冷冻保护剂,并研究其对猪精液冷冻效果的影响。结果表明:1.DMF(3%)对猪冷冻精子有一定的保护作用,效果略优于3%甘油(P＜0.05);2.DMF与甘油组成混合冷冻保护剂效果优于单一使用(P＜0.05),较佳总浓度和混合比例分别为3%和2∶1(V∶V)。
试验二,卵黄离心及精液透析预处理(不去除精浆)对猪精液冷冻效果影响的研究。在精液冷冻过程中,鸡蛋卵黄具有抗低温打击能力,可能是通过卵黄中的低密度脂蛋白(LDL)、磷脂及胆固醇来发挥保护作用。此外,卵黄中一些不规则的大颗粒物质容易在冷冻过程中形成大的冰晶,容易引起对精子的机械性损伤。而浑浊的稀释液也不利于精子分析仪对精子进行客观准确的评价;公猪精浆除了包含一些对精子有害的物质外,也有可能含有一些对精子起保护作用的成份,故其对精子在冷冻过程中的积极影响也不容忽视。本研究分别对卵黄进行离心处理,以及对包含精浆精液进行冻前透析处理,探索卵黄保护冷冻精子效果及精浆在降温平衡及冷冻过程中对精子的保护作用,从而实现优化现有方法与程序的目的。结果表明:对卵黄进行离心以及包含精浆精液采用透析处理均明显改善猪精子冷冻效果(P＜0.05)。
试验三,红景天多糖(Rhodiola Polysaccharide)对猪精液冷冻保护效果影响的研究。红景天多糖作为传统的中草药提取物,具有抗衰老、抗氧化和抗细胞凋亡等多种功能,尤其具有较强的清除超氧阴离子自由基(O~(2-))和抑制脂质过氧化作用的能力。然而,其作为冷冻保护剂用于细胞的冷冻保存却几乎未见报道。本试验在TCG冷冻稀释液的基础上,研究了红景天多糖对猪冷冻精子的保护作用,也对红景天多糖的不同添加方式对其保护效果进行了一些研究。结果表明:1.在猪精液预处理、冷冻过程中,红景天多糖能提供有效的抗氧化能力,红景天多糖较佳添加量为6mg/L;2.在Ⅰ液中添加红景天多糖效果优于在Ⅱ液中添加(P＜0.05),表明红景天多糖能在精液降温平衡过程中为猪精子提供有效的保护作用,较佳添加量为6mg/L。
试验四,大豆卵磷脂对猪精液冷冻保护效果影响的研究。大豆卵磷脂作为一种植物提取物,具有类似卵黄中具有抗低温打击保护能力的功能性成份。至今已证实大豆卵磷脂在牛等动物精液冷冻中有较好的保护作用,但在猪精液冷冻中却研究较少。本试验以20%卵黄作对照,研究了大豆卵磷脂替代卵黄的可行性以及其对猪精液冷冻保存的影响。结果表明:1.大豆卵磷脂对猪精液冷冻有较好的保护作用;2.较适宜的添加浓度为6%,其冻后精子活率、顶体完整率、质膜完整率及精子DNA完整性均明显高于卵黄对照组(P＜0.05)。
试验五,谷氨酰胺对猪精液冷冻保护效果影响的研究。氨基酸是生物功能大分子蛋白质的基本组成单位,也可以为细胞代谢提供一定的营养。谷氨酰胺在一些哺乳动物精液冷冻中发挥较好的保护作用,但在猪的精子冷冻中却很少采用。本试验在9%低密度脂蛋白(LDL)冷冻稀释液的基础上,研究不同浓度的谷氨酰胺对猪精液冷冻效果的影响。结果表明:LDL冷冻稀释液中添加谷氨酰胺可明显提高猪精子冷冻效果(P＜0.05),较适宜添加量为40 mM。
Research on the cryopreservation of boar spermatozoa was developed with the application of swine artificial insemination,and was thought as one important milestone in the history of swine industry.A "bank of boar spermatozoa" without the limitation of space-time by semen freezing could provide boar semen from an excellent boar for a lot of sows.In addition,the cryopreservation of boar spermatozoa also could provide a safeguard of biosafety for swine breeding.
It was well-known that there is still a big gap between the cryopreservation of boar spermatozoa and the cryopreservation of bovine spermatozoa.So the cryopreservation of boar spermatozoa was still not applied widely in swine industry for some time to come.
In order to improve the quality of frozen-thawed boar semen,two ways including freezing methods and freezing extenders were studied in this present study.The spermatozoa parameters(including spermatozoa motion),acrosome integrity,capacitated spermatozoa,membrane integrity,and DNA integrity were evaluated by computer-aided sperm analysis(CASA) system,Fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) staining,Chlortetracycline(CTC) staining,hypo-osmotic swelling test (HOST) and single cell gel electrophoresis(SCGE),respectively.
In this present study,five experiments were carried out.
Experiment 1,the effect of N,N-Dimethylformamide(DMF) on freezing boar spermatozoa.Previous studies showed that DMF could provide better protection for freezing spermatozoa than those of glycerol.However,few reports on freezing boar spermatozoa used DMF was found.We attempted to use a mixing cryoprotectant with DMF and glycerol in varing proportions.The results showed that the effect of freezing extender supplemented with 3%DMF was superior to those of freezing extender supplemented with 3%glycerol,and the mixed cryoprotectant(1%glycerol and 2%DMF) provided better protection for freezing boar spermatozoa than those of other treatment groups(P＜0.05).
Experiment 2,the effect of centrifugation of egg yolk and dialysis of boar semen during cooling process on freezing boar spermatozoa.Egg yolk was used to protect boar spermatozoa against cold shock due to Low density lipoproteins(LDL) and phospholipid of it's during cool process.Large particulate of egg yolk was thought as a disadvantage factor for cryopreservation and evaluation of spermatozoa motion by CASA system;the role of boar semen plasma should not be neglected due to its property comparing with other domestic animals.So we centrifuged the egg yolk and dialyzed boar semen during cool process to remove the large particulate and the small molecules respectively which was harmful to spermatozoa comparing with the control treatments.Results: centrifugation of egg yolk and dialysis of boar semen during cool process could improve the effect on freezing boar spermatozoa comparing the control treatments respectively (P＜0.05).However,further studies are needed to reduce its cost and complexity.
Experiment 3,the protective effect of rhodiola polysaccharide on freezing boar spermatozoa.As aqueous extract from Chinese Herb,rhodiola polysaccharide had strong scavenging activity against superoxide anion radical(O~(2-)) and inhibited lipid peroxidation. However,there are apparently no published data regarding the use of to prevent formation of ROS during cryopreservation of boar spermatozoa.Based on TCG extender, the effect of different concentrations or methods of rhodiola polysaccharide on freezing boar spermatozoa was evaluated by previous test methods and enzyme assay.Results:1. rhodiola polysaccharide could provide better antioxidant protection for boar spermatozoa during cooling and thaw-thawed process;2.the effect of rhodiola polysaccharide added inⅠextender was better than added inⅡextender and the proper concentration of it was 6 mg/L.
Experiment 4,the effect of soy lecithin on freezing boar spermatozoa.Soy lecithin extracting from Soybean was similar with the functional ingredients of egg yolk.It was reported that freezing extender containing soy lecithin could provide better protection for bovine spermatozoa.However,few relative reports on boar were found.We investigated the effect of soy lecithin on cryopreserved boar semen compared with the treatments with 20%egg yolk.Results:1.Soy lecithin could provide better protection than that of 20% egg yolk;2.The proper concentration of soy lecithin was 6%,and the motility,the percentage of acrosome integrity,membrane integrity and DNA integrity were higher than those of 20%egg yolk treatment(P＜0.05).
Experiment 5,the effect of glutamine on on freezing boar spermatozoa.Amino acids are the basic components of proteins and can provide energy for cellular metabolism in vivo.Studies have shown that,as one of Amino acids,glutamine could provide better protection for freezing spermatozoa of domestic animals.However,few reports on boar spermatozoa were found.Based on 9%LDL extender,the effect of different concentrations of glutamine on freezing boar spermatozoa was tested in this present study. Results:glutamine could provide better protection for boar spermatozoa during cryopreservation,and the proper concentration was 40 mM.
为了进一步改善猪冷冻精子的品质,本研究在改进猪精液冷冻方法与优化冷冻稀释液两个方面进行了研究,以精子活率、线粒体活性、精子质膜完整性、精子项体完整率和低渗膨胀测试(HOST)等为指标,利用精子自动分析仪(CASA)结合荧光染色方法及一些常规检测手段对精子品质进行了客观有效的评价。
本论文主要针对以下五个方面进行研究:
试验一,N,N-二甲基甲酰胺(DMF)提高猪精液冷冻效果研究。近几年来,一些研究者将DMF用于家畜精子冷冻中,在一些家畜精液冷冻中进行了尝试,并取得优于甘油的效果。然而,DMF在猪精液冷冻中的研究却很少报道。本试验以DMF和甘油组成混合冷冻保护剂,并研究其对猪精液冷冻效果的影响。结果表明:1.DMF(3%)对猪冷冻精子有一定的保护作用,效果略优于3%甘油(P＜0.05);2.DMF与甘油组成混合冷冻保护剂效果优于单一使用(P＜0.05),较佳总浓度和混合比例分别为3%和2∶1(V∶V)。
试验二,卵黄离心及精液透析预处理(不去除精浆)对猪精液冷冻效果影响的研究。在精液冷冻过程中,鸡蛋卵黄具有抗低温打击能力,可能是通过卵黄中的低密度脂蛋白(LDL)、磷脂及胆固醇来发挥保护作用。此外,卵黄中一些不规则的大颗粒物质容易在冷冻过程中形成大的冰晶,容易引起对精子的机械性损伤。而浑浊的稀释液也不利于精子分析仪对精子进行客观准确的评价;公猪精浆除了包含一些对精子有害的物质外,也有可能含有一些对精子起保护作用的成份,故其对精子在冷冻过程中的积极影响也不容忽视。本研究分别对卵黄进行离心处理,以及对包含精浆精液进行冻前透析处理,探索卵黄保护冷冻精子效果及精浆在降温平衡及冷冻过程中对精子的保护作用,从而实现优化现有方法与程序的目的。结果表明:对卵黄进行离心以及包含精浆精液采用透析处理均明显改善猪精子冷冻效果(P＜0.05)。
试验三,红景天多糖(Rhodiola Polysaccharide)对猪精液冷冻保护效果影响的研究。红景天多糖作为传统的中草药提取物,具有抗衰老、抗氧化和抗细胞凋亡等多种功能,尤其具有较强的清除超氧阴离子自由基(O~(2-))和抑制脂质过氧化作用的能力。然而,其作为冷冻保护剂用于细胞的冷冻保存却几乎未见报道。本试验在TCG冷冻稀释液的基础上,研究了红景天多糖对猪冷冻精子的保护作用,也对红景天多糖的不同添加方式对其保护效果进行了一些研究。结果表明:1.在猪精液预处理、冷冻过程中,红景天多糖能提供有效的抗氧化能力,红景天多糖较佳添加量为6mg/L;2.在Ⅰ液中添加红景天多糖效果优于在Ⅱ液中添加(P＜0.05),表明红景天多糖能在精液降温平衡过程中为猪精子提供有效的保护作用,较佳添加量为6mg/L。
试验四,大豆卵磷脂对猪精液冷冻保护效果影响的研究。大豆卵磷脂作为一种植物提取物,具有类似卵黄中具有抗低温打击保护能力的功能性成份。至今已证实大豆卵磷脂在牛等动物精液冷冻中有较好的保护作用,但在猪精液冷冻中却研究较少。本试验以20%卵黄作对照,研究了大豆卵磷脂替代卵黄的可行性以及其对猪精液冷冻保存的影响。结果表明:1.大豆卵磷脂对猪精液冷冻有较好的保护作用;2.较适宜的添加浓度为6%,其冻后精子活率、顶体完整率、质膜完整率及精子DNA完整性均明显高于卵黄对照组(P＜0.05)。
试验五,谷氨酰胺对猪精液冷冻保护效果影响的研究。氨基酸是生物功能大分子蛋白质的基本组成单位,也可以为细胞代谢提供一定的营养。谷氨酰胺在一些哺乳动物精液冷冻中发挥较好的保护作用,但在猪的精子冷冻中却很少采用。本试验在9%低密度脂蛋白(LDL)冷冻稀释液的基础上,研究不同浓度的谷氨酰胺对猪精液冷冻效果的影响。结果表明:LDL冷冻稀释液中添加谷氨酰胺可明显提高猪精子冷冻效果(P＜0.05),较适宜添加量为40 mM。
Research on the cryopreservation of boar spermatozoa was developed with the application of swine artificial insemination,and was thought as one important milestone in the history of swine industry.A "bank of boar spermatozoa" without the limitation of space-time by semen freezing could provide boar semen from an excellent boar for a lot of sows.In addition,the cryopreservation of boar spermatozoa also could provide a safeguard of biosafety for swine breeding.
It was well-known that there is still a big gap between the cryopreservation of boar spermatozoa and the cryopreservation of bovine spermatozoa.So the cryopreservation of boar spermatozoa was still not applied widely in swine industry for some time to come.
In order to improve the quality of frozen-thawed boar semen,two ways including freezing methods and freezing extenders were studied in this present study.The spermatozoa parameters(including spermatozoa motion),acrosome integrity,capacitated spermatozoa,membrane integrity,and DNA integrity were evaluated by computer-aided sperm analysis(CASA) system,Fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) staining,Chlortetracycline(CTC) staining,hypo-osmotic swelling test (HOST) and single cell gel electrophoresis(SCGE),respectively.
In this present study,five experiments were carried out.
Experiment 1,the effect of N,N-Dimethylformamide(DMF) on freezing boar spermatozoa.Previous studies showed that DMF could provide better protection for freezing spermatozoa than those of glycerol.However,few reports on freezing boar spermatozoa used DMF was found.We attempted to use a mixing cryoprotectant with DMF and glycerol in varing proportions.The results showed that the effect of freezing extender supplemented with 3%DMF was superior to those of freezing extender supplemented with 3%glycerol,and the mixed cryoprotectant(1%glycerol and 2%DMF) provided better protection for freezing boar spermatozoa than those of other treatment groups(P＜0.05).
Experiment 2,the effect of centrifugation of egg yolk and dialysis of boar semen during cooling process on freezing boar spermatozoa.Egg yolk was used to protect boar spermatozoa against cold shock due to Low density lipoproteins(LDL) and phospholipid of it's during cool process.Large particulate of egg yolk was thought as a disadvantage factor for cryopreservation and evaluation of spermatozoa motion by CASA system;the role of boar semen plasma should not be neglected due to its property comparing with other domestic animals.So we centrifuged the egg yolk and dialyzed boar semen during cool process to remove the large particulate and the small molecules respectively which was harmful to spermatozoa comparing with the control treatments.Results: centrifugation of egg yolk and dialysis of boar semen during cool process could improve the effect on freezing boar spermatozoa comparing the control treatments respectively (P＜0.05).However,further studies are needed to reduce its cost and complexity.
Experiment 3,the protective effect of rhodiola polysaccharide on freezing boar spermatozoa.As aqueous extract from Chinese Herb,rhodiola polysaccharide had strong scavenging activity against superoxide anion radical(O~(2-)) and inhibited lipid peroxidation. However,there are apparently no published data regarding the use of to prevent formation of ROS during cryopreservation of boar spermatozoa.Based on TCG extender, the effect of different concentrations or methods of rhodiola polysaccharide on freezing boar spermatozoa was evaluated by previous test methods and enzyme assay.Results:1. rhodiola polysaccharide could provide better antioxidant protection for boar spermatozoa during cooling and thaw-thawed process;2.the effect of rhodiola polysaccharide added inⅠextender was better than added inⅡextender and the proper concentration of it was 6 mg/L.
Experiment 4,the effect of soy lecithin on freezing boar spermatozoa.Soy lecithin extracting from Soybean was similar with the functional ingredients of egg yolk.It was reported that freezing extender containing soy lecithin could provide better protection for bovine spermatozoa.However,few relative reports on boar were found.We investigated the effect of soy lecithin on cryopreserved boar semen compared with the treatments with 20%egg yolk.Results:1.Soy lecithin could provide better protection than that of 20% egg yolk;2.The proper concentration of soy lecithin was 6%,and the motility,the percentage of acrosome integrity,membrane integrity and DNA integrity were higher than those of 20%egg yolk treatment(P＜0.05).
Experiment 5,the effect of glutamine on on freezing boar spermatozoa.Amino acids are the basic components of proteins and can provide energy for cellular metabolism in vivo.Studies have shown that,as one of Amino acids,glutamine could provide better protection for freezing spermatozoa of domestic animals.However,few reports on boar spermatozoa were found.Based on 9%LDL extender,the effect of different concentrations of glutamine on freezing boar spermatozoa was tested in this present study. Results:glutamine could provide better protection for boar spermatozoa during cryopreservation,and the proper concentration was 40 mM.
引文
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