結合甘油和低密度脂蛋白冷凍保護劑與公豬精漿解凍劑對解凍公豬精子質量之影響

Effect of Glycerol and Low-Density Lipoprotein Cryoprotectants Combined with Boar Seminal Plasma Thawing Agent in Thawed Boar Sperm Quality

譚蔚詩(Tam Wai-Sze)

指導教授 ： 彭劭于 ； 林聖淇

國立屏東科技大學/國際學院/熱帶農業暨國際合作系/碩士(2020年)

https://doi.org/10.6346/NPUST202000267

摘要

冷凍解凍後精子之活動力及存活率相較於新鮮精液會大幅降低，其原因為冷凍過程精子細胞發生氧化作用及水分結晶，導致解凍過程細胞死亡。甘油與低密度脂蛋白 ( LDL ) 是最常見組合之冷凍保護液，為了找出提高解凍後杜洛克混高畜黑豬品種 ( DK ) 公豬精子品質之方法，試驗甘油、LDL及精漿，評估最適合作精子冷凍保護劑，鏡檢評估精子活力及利用SYBR-Green I和PI作螢光染色或伊紅-苯胺黑 ( Eosin-Nigrosin ) 計算存活率及精子異常形態。試驗一結果顯示添加4%和6%甘油冷凍保護劑對精子活動力顯著較添加0%、2%、8%甘油者為高 ( 27.0% ± 1.3、24.2 ± 1.9 v.s. 10.4% ± 1.8、8.0% ± 1.4、17.6% ± 2.3 ) ( P < 0.05 )。此外，添加4%甘油冷凍保護劑對精子存活率顯著較添加0%、2%、6%、8%甘油者為高 ( 38.8% ± 5.8 v.s. 5.7 % ± 2.5、22.5% ± 4、26.7% ± 2.3、16.2% ± 4.8 ) ( P < 0.05 )。再者，試驗結果顯示添加4%甘油冷凍保護劑對精子異常形態顯著 ( P < 0.05 ) 較添加6%甘油者為低 ( 8.2% ± 2.4 v.s. 22.2% ± 7.0 )。試驗二是比較添加不同濃度之LDL成份對精子品質之影響，試驗二結果顯示添加LDL冷凍保護劑對精子活動力顯著 ( P < 0.05 ) 較對照組 ( 蛋黃+甘油 ) 者低 ( 11.6% ± 3.1、13.4% ± 3.8、13.7% ± 3.7 v.s. 27.0% ± 1.3 )。添加8%和9%LDL冷凍保護劑對精子存活率顯著高於 ( P < 0.05 ) 對照組及10%LDL ( 58.2% ± 4.9、61.1% ± 5.5 v.s. 38.8% ± 5.6、45.0% ± 2.4 )。試驗三是分析評估添加不同濃度之精漿於精液解凍液中比較解凍精子之品質。試驗三結果顯示對照組 ( 蛋黃+甘油 ) 冷凍保護劑對精子活動力顯著 ( P < 0.05 ) 較添加25%、50%、75%、100%精漿者高 ( 13.4% ± 3.8 v.s. 5.8% ± 1.3、9.0% ± 1.8、8.1% ± 2.3、6.5% ± 1.7 )。對照組、25%和50%精漿冷凍保護劑對精子存活率顯著 ( P < 0.05 ) 較75%及100%精漿者高 ( 61.1% ± 5.5、59.8% ± 1.9、65.1% ± 4.0 v.s. 50.7% ± 4.3、40.8% ± 8.2 )藉此研究得出使用4%甘油、8%~9%LDL冷凍保護劑能有效提升冷凍解凍後之DK公豬精子品質。

關鍵字

DK公豬精子 ； 冷凍解凍精子 ； 冷凍保護劑 ； 甘油 ； 低密度脂蛋白 ； 精漿

並列摘要

Compared with fresh semen, the motility and viability of semen after freezing and thawing are greatly reduced. The main causes are oxidation of sperm cells during freezing, water crystals in sperm cells and cell death due to cell rupture during thawing. Glycerol and low-density lipoprotein ( LDL ) are the most common combination of cryoprotectants. In order to find ways to improve the quality of thawed sperm, this report will analyze the glycerol, LDL and seminal plasma ( SP ) combinations of different concentrations and evaluate the most suitable cryopreservation agent for Duroc x KHAPS Black Pig ( DK ) boar sperm, to assessed sperm motility by microscopy, and calculated the viability and percentage of abnormal sperm morphology by using SYBR-Green I and PI for fluorescent staining or eosin-nigrosin. The results showed that the viability of sperm with 4% glycerin was 38.8±5.8, significantly different ( P < 0.05 ) from that of other groups ( 0%, 2%, 6% and 8% glycerol ), while the motility of thawed sperm with 4% glycerol was 27.0 ± 1.3, and that of thawed sperm with 6% glycerol was 24.2 ± 1.9, significantly different ( P < 0.05 ) from that of other groups ( 0%, 8% glycerol ). The second experiment is to analyze and compare adding different concentration of LDL composition of defrosted semen quality, add the concentration of LDL after thawing motility was significantly lower ( P < 0.05 ) than the control group 27.0 ± 1.3 ( 4% glycerol + egg yolk ), add thawed sperm viability of 8% and 9% of LDL were 58.2 ± 4.9 and 61.1 ± 5.5 respectively, they are significantly higher ( P < 0.05 ) than other group ( control group and add 10% LDL ). The last group of experiments was to evaluate the quality of thawed sperm by adding different concentrations of SP into the semen thawed liquid. The result showed that the cryoprotectant in the control group (yolk + glycerol) had a significant effect (P < 0.05) on sperm motility compared with those with 25%, 50%, 75% and 100% SP. Compared with the percentage of abnormal morphology, there was a significant difference ( P < 0.05 ) between the thawed sperm of 75% SP got 24.5 ± 2.8 and 25% SP got 13.8 ± 1.5. From this study, it was found that using 4% glycerol, 8% to 9%LDL could effectively improve the quality of frozen thawed DK boar sperm.

並列關鍵字

DK boar sperm ； semen cryopreservation ； cryoprotectants ； glycerol ； low-density lipoprotein ； seminal plasma

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