本文關(guān)鍵詞： 豬精液 鞣花酸 常溫保存 氧自由基 冷打擊　出處：《西北農(nóng)林科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：豬的人工授精技術(shù)是豬規(guī)�；B(yǎng)殖的關(guān)鍵技術(shù)之一,而精液保存質(zhì)量則是保證人工授精成功率與母豬產(chǎn)仔數(shù)的關(guān)鍵因素之一。豬精液體外保存的方式有三種:即常溫保存、低溫和冷凍保存。由于豬精子細(xì)胞膜的膽固醇/磷脂較低,精子對于溫度相對敏感,對低溫的耐受能力較差,因此,在生產(chǎn)實踐中多采用豬精液常溫保存技術(shù)保存精液。然而,在常溫保存過程中精子細(xì)胞內(nèi)會產(chǎn)生過量的氧自由基(ROS),對精子質(zhì)膜和頂體完整性造成氧化損傷,使精子的運動能力和活力降低,導(dǎo)致精液保存品質(zhì)和人工授精成功率降低。為了降低ROS對于精子的損傷,提高精液保存質(zhì)量,為此本研究向豬精液常溫保存稀釋液Modena中添加了一種天然植物提取物抗氧化劑——鞣花酸。本研究利用計算機輔助精子質(zhì)量分析儀(CASA)、熒光顯微鏡、多功能酶標(biāo)儀等儀器,檢測鞣花酸對大白豬精液常溫保存效果的影響并探究其作用途徑;同時研究了鞣花酸對于豬精子冷打擊的保護作用;以期減緩豬精液保存過程中產(chǎn)生的氧化應(yīng)激、延長精液有效保存時間,同時為我國精液保存抗氧化劑的研發(fā)提供科學(xué)資料。本研究獲得如下重要結(jié)果:1.將Modena稀釋液分為6組分別添加不同濃度的鞣花酸,1:1稀釋精液(鞣花酸終濃度為:0、15、25、35、50、75μmol/L),并檢測保存1、3、5、7、9 d的精子活、質(zhì)膜及頂體的完整情況,結(jié)果發(fā)現(xiàn):15、25μmol/L的鞣花酸均能提高精子的保存質(zhì)量;而且,當(dāng)鞣花酸添加濃度為25μmol/L時,保存7d的精子活力為66.63%,質(zhì)膜完整率為84.05%、頂體完整率為87.07%,均顯著優(yōu)于其他處理組(P0.05);精子的快速前進(jìn)行性運動率(PRO)、平均直線運動速率(VSL)和精子平均路徑速度(VAP)顯著高于對照組(P0.05);此外,精子平均曲線運動速率(VCL)與對照組相比無顯著差異(P0.05)。這些結(jié)果表明,鞣花酸對于豬精液常溫保存具有保護作用,最佳添加濃度為25μmol/L。2.檢測常溫保存7d后精子細(xì)胞內(nèi)ROS、脂質(zhì)過氧化水平和總抗氧化能力,發(fā)現(xiàn)25μmol/L鞣花酸處理組的ROS和脂質(zhì)過氧化水平最低,總抗氧化能力最高,且與對照組相比結(jié)果顯著(P0.05)。提示鞣花酸能夠通過清除精子細(xì)胞內(nèi)ROS、降低脂質(zhì)過氧化水平和提高總抗氧化能力的途徑來保護精子,緩解常溫保存過程中精子的氧化損傷。3.在豬精子由體溫快速降溫至5℃的過程中,發(fā)現(xiàn)豬精子活力大幅下降,在精子遭受冷打擊時,鞣花酸對精子活力的影響與對照組相比結(jié)果不顯著,表明鞣花酸保護豬精子免受冷打擊損傷的作用不顯著。綜上,本研究證明在豬精液常溫保存過程中添加鞣花酸能夠通過清除精子細(xì)胞內(nèi)的活性氧自由基,降低脂質(zhì)過氧化水平和提高總抗氧化能力等作用途徑,來提高大白豬精液常溫保存的質(zhì)量,且在豬精液稀釋液Modena的最佳添加濃度為25μmol/L。此外,當(dāng)精子遭受冷打擊時,鞣花酸對其保護結(jié)果不顯著。
[Abstract]:Artificial insemination is one of the key techniques in large-scale pig breeding, and the quality of semen preservation is one of the key factors to ensure the success rate of artificial insemination and the litter size of sows. Cryopreservation and cryopreservation. Because of the low cholesterol / phospholipid ratio of porcine sperm cell membrane, the sperm is sensitive to temperature and has poor tolerance to low temperature. During normal temperature preservation, excessive oxygen free radicals will be produced in sperm cells, which will cause oxidative damage to the integrity of sperm plasma membrane and acrosome, and decrease the motility and motility of spermatozoa. In order to reduce the damage of ROS to sperm and improve the quality of semen preservation, In this study, a natural plant extract antioxidant, tannic acid, was added to porcine semen at room temperature preservation diluent (Modena). A computer-assisted sperm quality analyzer (CASAA), fluorescence microscope, and multifunction enzyme marker were used in this study. The effects of tannic acid on cryopreservation of pig semen at room temperature and the protective effect of tannic acid on cold attack of pig sperm were studied in order to slow down the oxidative stress during the preservation of pig semen. Prolong the effective preservation time of semen, At the same time, it provided scientific data for the research and development of antioxidants for semen preservation in China. In this study, the following important results were obtained: 1: 1.The diluents of Modena were divided into 6 groups, which were diluted with different concentrations of tannic acid and 1: 1 respectively. The sperm motility was detected after the storage of 1 ~ (3) C ~ (5) C _ (7) C ~ (2 +) for 9 days. The integrity of plasma membrane and acrosome was obtained. The results showed that tannic acid at 25 渭 mol/L could improve the quality of spermatozoa, and when the concentration of tannic acid was 25 渭 mol/L, the quality of spermatozoa could be improved when the concentration of tannic acid was 25 渭 mol/L. The sperm motility, plasma membrane integrity and acrosomal integrity were 66.63%, 87.07%, 87.07% and 87.07%, respectively, which were significantly better than those of other treatment groups (P 0.05%), and the rate of progressive sperm motility before rapid progression was significantly higher than that of the other treatment groups (P < 0.05). The average linear motion rate (VSLL) and the average sperm path velocity (VAPs) were significantly higher than those in the other treatment groups. In addition, in the control group, P0.05, There was no significant difference in sperm mean curve motility rate (VCLL) compared with the control group (P 0.05). These results showed that tannic acid had protective effect on the preservation of porcine semen at room temperature. The best addition concentration was 25 渭 mol / L ~ (-2). After 7 days of storage at room temperature, ros, lipid peroxidation and total antioxidant capacity of spermatozoa were measured. The results showed that 25 渭 mol/L tannic acid treatment group had the lowest level of ROS and lipid peroxidation, and the highest total antioxidant capacity. Compared with the control group, the results showed that tannin could protect spermatozoa by scavenging ROSin, decreasing lipid peroxidation and increasing total antioxidant capacity. Alleviating oxidative damage of spermatozoa during normal temperature preservation. During the rapid cooling of porcine spermatozoa from body temperature to 5 鈩，

本文編號：1553381