本土黑豬冷凍精液之製作及解凍後人工授精之懷孕率研究

Study on the production method of domestic Taiwan black boar frozen semen and artificial insemination on black sow

陳紀良(Chen Chi-Leung)

指導教授 ： 彭劭于 ； 沈朋志 ； 楊國泰

國立屏東科技大學/農學院/動物科學與畜產系所/碩士(2022年)

https://doi.org/10.6346/NPUST202200421

摘要

本土黑豬屬於台灣民間品種，為了保留原品種特有之性狀，故冷凍精液之製作方式極為重要。豬隻精液非常敏感，解凍後的冷凍精液活力通常不佳，一般影響精子活力及品質之因素包含：降溫速率、冷凍保護劑、麥管容量及抗氧化劑等。
因此本研究將利用不同製作方式來製作冷凍精液，並解凍精液觀察精子的表現及實施人工授精。沿用前人的冷凍精液製作方式，分為保溫階段（holding time）與平衡階段（equilibration），最後填裝麥管後，放置在液態氮上冷凍，再投入液態氮中直接進行保存；冷凍保護劑分別使用低密度脂蛋白（Low density lipoprotein, LDL）、離心蛋黃與新鮮蛋黃來保護精子減少在冰晶形成時細胞型態被破壞，並添加不同抗氧化劑來減少細胞被氧化。本試驗將對於不同製作方式與不同的冷凍保護劑及抗氧化劑的添加差異進行比較，並進一步對精子之活力及存活率表現做觀察。
研究結果顯示甘油比例，添加5%與6%甘油的精子活力與存活率有顯著差異（p < 0.05），冷卻速率使用階段式降溫能有效提升精子的活力與存活率（p < 0.01）；冷凍保護劑使用LDL或新鮮蛋黃皆優於離心蛋黃；冷凍精液放置於液態氮液面高度3 cm，解凍時利用50℃－12秒解凍冷凍精液其活力優於液態氮液面高度1 cm者（p < 0.05）；冷凍精液放置於液態氮液面高度3 cm及5 cm，解凍時利用50℃－12秒解凍冷凍精液，其活力優於液態氮液面高度1 cm及5 cm，與利用37℃－20秒、70℃－8秒解凍之組合；添加紫錐花萃取物於冷凍精液冷凍解凍精液，活力沒有顯著差異，但是在存活率添加0.1、0.2、及0.3%紫錐花萃取物皆優於無添加組別（p < 0.05）；於冷凍精液解凍進行人工授精之試驗結果中，比較其懷孕率新鮮精液（90%）優於冷凍精液（56%）。為了避免台灣原生品種及種原因生長性能不符效益而逐漸被淘汰，或因重大疫病而撲殺種畜，造成重要豬隻遺傳資源的流失，不斷地改良以獲取更佳的冷凍精液製作方式與條件，對於台灣本土黑豬種原活化運用極其重要，技術的提升不僅是能夠加深產業應用，更是確保珍貴種原永續存在重要的一環。

關鍵字

紫錐花萃取物 ； 冷凍精液 ； 低密度脂蛋白 ； 活力 ； 平埔黑豬

並列摘要

Domestic black boars in Taiwan are considered a unique species and in order to retain the unique characteristics of the original breed, the methods of freezing its semen is extremely important. Boar semen is very sensitive, and the frozen semen post thawed usually not in a good motility condition. Generally, factors that affect the sperm's motility and quality include: cooling rate, cryoprotectant, straw capacity and antioxidants, etc.
Therefore, this study will use different production methods to observe the performance of sperm, and frozen-thawed semen for artificial insemination. Using the predecessor's method of freezing black boar semen, it is divided into two stages: holding time and equilibration. After the straw is filled, it is placed ontop of liquid nitrogen for freezing, then put into liquid nitrogen for direct preservation. Cryoprotectants that have been previously used by predecessor are low density lipoprotein (LDL), centrifuged egg yolk and fresh egg yolk to protect sperm and reduce cell morphology damage when water crystals are formed, and different antioxidants have been added to reduce cell oxidation. In this experiment, the differences in freezing rate, glycerol ratio, Echinacea extract, liquid level and frozen-thawed temperature will be compared, and further the differences in sperm motility and viability will be discuss.
Additional of 5% and 6% glycerol significantly increases sperm viability (p < 0.05). The results show that stage cooling method can effectively improve sperm motility and viability (p < 0.01).LDL or fresh egg yolk as cryoprotectant is better than centrifuged egg yolk. The frozen semen placed ontop of liquid nitrogen height of 3 cm and 5 cm combine with thawing method 50°C for 12 seconds was better than that of liquid nitrogen at a height of 1 cm and 5 cm. 37℃ for 20 seconds, 70℃ for 8 seconds motility and viability (p < 0.05). The addition of Echinacea extract in frozen-thawed semen was no significant difference in motility. However the viability of adding 0.1%, 0.2%, and 0.3% Echinacea extract better than (p < 0.05) control group and post thawing semen artificial insemination in sows, the pregnancy rate of fresh semen(90%) is better than post thawing semen(56%). In order to avoid the gradual elimination of Domestic Taiwan black boars breeds and breeders due to their inconsistent growth performance, resulting in the loss of important pig genetic resources, continuous improvement has been made to obtain better frozen semen production methods and conditions. Important, the improvement of technology is not only able to deepen industrial applications, but also an important part of ensuring the sustainable existence of precious seeds.

並列關鍵字

Echinacea extract ； Cryopreservation ； Low-density lipoprotein ； Motility ； Pinpu boar

參考文獻

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