本文選題：豬 + 精液冷凍��； 參考：《上海海洋大學》2015年碩士論文

【摘要】：低溫冷凍解凍使液氮保存的豬精子細胞發(fā)生嚴重的凋亡,常見的凋亡途徑——死亡受體外源性途徑和線粒體內(nèi)源性途徑均可能在凍后精子中發(fā)生,介導凍融精子發(fā)生凋亡而降低精子質(zhì)量。本實驗在實驗室之前研究基礎(chǔ)上,通過對凋亡途徑相關(guān)基因的表達情況研究探索凋亡發(fā)生的可能相關(guān)途徑,并探索在冷凍保護劑中添加抗氧化劑白藜蘆醇對冷凍解凍后豬精子細胞凋亡和凋亡途徑的影響。研究結(jié)果:1.冷凍精子TUNEL染色后的凋亡率(80.4%)與新鮮組(9.7%)相比顯著升高(P0.05);與新鮮組精子內(nèi)ROS綠色熒光比率相比,冷凍組(58.2%)亦顯著高于新鮮組(P0.05);與新鮮組0.926μmol/L的ATP濃度相比,冷凍組精子的ATP濃度(0.247μmol/L)則顯著下降(P0.05);q RT-PCR檢測結(jié)果顯示,冷凍組中TNF-α、Fas、Caspase家族、P53和Bax等基因相對表達量升高,其中Fas、P53、TNF-α、caspase 8和caspase 9等相對表達量升高顯著(P0.05);BCL-2、BIRC-5、Mn SOD、Cu Zn SOD和SURVIVN等基因相對表達量降低,其中BIRC-5、Mn SOD、SURVIVN降低顯著(P0.05)。2.向冷凍劑中添加白藜蘆醇工作液,使其在冷凍劑中的終濃度分別為12.5μM,25μM,50μM和100μM,并設(shè)置0μM作為空白對照,比較各組凍融精子活率,頂體和質(zhì)膜完整情況。結(jié)果表明白藜蘆醇終濃度為50μM組的精子活力,質(zhì)膜完整率和頂體完整率均顯著高于其余各添加組(P0.05),并顯著高于0μM組的精子質(zhì)量。精子各指標隨著白藜蘆醇濃度增大而升高,呈現(xiàn)濃度依賴性,但當白藜蘆醇濃度過高(≥100μM)時精子質(zhì)量顯著下降(P0.05)。3.凍融精子經(jīng)Caspase原位熒光染色后,添加白藜蘆醇組的實驗組凍融精子凋亡率為29.0%,與未添加白藜蘆醇組的對照組(86.3%)相比顯著降低(P0.05);實驗組58.6%的ROS綠色熒光比率亦顯著低于對照組(88.4%)(P0.05);與對照組精子m RNA各基因相比,caspase-8相對表達量降低,Fas,P53,TNF-α,Bax,caspase-3,caspase-9相對表達量顯著降低(P0.05);Cu Zn SOD相對表達量升高,Bcl-2,Mn SOD,SURVIVN等顯著升高(P0.05)。
[Abstract]:Cryopreservation and thawing caused severe apoptosis of porcine spermatozoa. The common apoptotic pathway, the exogenous pathway of death receptor and the endogenous pathway of mitochondria, may occur in frozen spermatozoa. Mediated apoptosis of freeze-thawed spermatozoa and reduced sperm quality. On the basis of pre-laboratory studies, this study explored the possible pathway of apoptosis through the study of the expression of genes related to the apoptosis pathway. The effects of anti-oxidant resveratrol on the apoptosis and apoptosis of porcine spermatozoa after freezing and thawing were explored. The result of the study was: 1. The apoptotic rate of frozen spermatozoa after TUNEL staining was significantly higher than that of fresh group (P 0.05), the green fluorescence ratio of ROS in frozen sperm was significantly higher than that in fresh group (P 0.05), and the concentration of ATP in fresh group was 0.926 渭 mol/L higher than that in fresh group. The ATP concentration of spermatozoa in freezing group was 0.247 渭 mol / L. The results showed that the relative expression of TNF- 偽 Fas-Caspase family p53 and Bax in frozen group was higher than that in control group. The relative expressions of Fas-P53, TNF- 偽 and caspase _ 9 increased significantly, and the relative expression of BCL-2BIRC-5Mn-SODN, Cu, Zn SOD and SURVIVN were significantly decreased, and BIRC-5Mn-SOD- SURVIVN decreased significantly (P0.05.2UVIVN). The final concentration of resveratrol was 12.5 渭 m, 25 渭 M, 50 渭 M and 100 渭 M, respectively. 0 渭 M was used as blank control to compare the viability of frozen and thawed spermatozoa and the integrity of acrosome and plasma membrane. The results showed that the sperm motility, plasma membrane integrity and acrosome integrity were significantly higher in the group of 50 渭 M resveratrol than those in the other groups, and the sperm quality was significantly higher than that in the 0 渭 M group. The sperm parameters increased with the increase of resveratrol concentration in a concentration-dependent manner, but the sperm quality decreased significantly when the concentration of resveratrol was too high (鈮，

本文編號：1874169