猪精液之冷冻保存

猪精液之冷冻保存 豬精液之冷凍保存 Cryopreservation of Boar Semen 研究生,辜煌輝 Ku, Huang-Hui 指導教授,謝來安 Hsieh, Lai-An 【摘要】 豬精液冷凍的優點為使用時較不受時間、地域的限制,因而提高優良種公豬的利用率,減少飼養種畜的開支且加速品種改良,另外也具有保種之意義,同時也是提供學術研究之方便性。本研究之目的為欲改良冷凍稀釋液的成分、調整冷凍解凍速率,以及探討一般稀釋液對精液稀釋的影響,希望透過這些研究可改善精液冷凍及解凍後之效果。結果顯示,新鮮精液稀釋倍數以1: 1稀釋時,不論是在15?或39?保存時,對於精子活動力及未染上Trypan blue的精子比率,有較1: 0、1: 2、1: 4及1: 10等稀釋倍率處理組者為優。在不同清潔劑種類與濃度對於精子活動力影響的試驗中,發現含1,、2,和4,Tween 20及Tween 80並不適合添加至冷凍稀釋液中。添加高濃度的清潔劑,第一階段與第二階段均添加1.5,OEP,在5?長期的儲存對精子的活動力是不利的,但是如添加後短時間內即製作冷凍精液,對於解凍後精子活動力與其它處理組並無顯著差異。在第一階段與第二階段冷凍稀釋液之蛋黃稀釋液是否經過離心,對於解凍後精子的活動力以及未染上Trypan blue精子比率均無影響。在第一階段冷凍稀釋液如添加20 mg/ml taurine或20 mg/ml L-glutamine,對解凍後精子活動力沒有改善。在製作冷凍精液過程中,將含有冷凍稀釋液精液放入-20?的冰箱以不同的時間降溫,5-15 min,,然後再放入保力龍盒裡受液氮燻煙不同時間,5-20 min,,解凍後所得結果與對照組,傳統液氮蒸燻20分鐘,無顯著差異,如在-20?冰箱冷卻25分鐘後立即擲入液氮中,解凍後其精子活動力降為0,而放置於-70?冰箱降溫25 min後投入液態氮中,則與對照組,傳統液氮蒸燻20分鐘,無顯著差異。在解凍速率方面,對照組與置於-70?冰箱20 min或1 hr,再置於50?水浴槽35 sec或 40 sec解凍之處理組間,對精子活動力與未染上Trypan blue比率並沒有顯著差異。在冷凍前新鮮精液經由MIII,Kiev,、Androhep、BTS和Modena等不同稀釋液處理對精液冷凍解凍後其精子之表現,Modena稀釋液之精子活動力顯著的低於其它各組,而解凍後精液以上述稀釋液分別稀釋,剛解凍時MIII,Kiev,稀釋液組顯著的低於其他各組,3 hr後Modena稀釋液組顯著的優於其它各組。在冷凍精液授精試驗中,解凍後具有較高精子活動力者,其受胎率較高。 關鍵字,公豬、冷凍保存、精子活動力、精液、稀釋液、Trypan blue、Kiev、Modena 【Abstract】 Major advantages of using frozen boar semen are as follows: not limited by geographic or time limitation, genetic improvement, economical service and for animal preservation. The objective of this study was to investigate the influence of semen extenders, frozen-thawed speed rate and egg-yolk component on motility of frozen-thawed spermatozoa. Results of the study indicated that the motility and trypan blue unstained in 1:1 dilution were higher than other groups (1:0, 1:2, 1:4 and 1:10). Tween 20 and Tween 80 were shown not good additives for freeze extender. If boar semen diluted with extender supplement with high concentration detergent (1st and 2nd with 1.5% OEP) and stored at 5? for over 24 hr, the sperm motility was low. However, there is not disadvantageous if semen store for a short time. Either egg-yolk extender go through centrifugation or not, there were no significant difference among sperm motility and trypan blue unstained after frozen-thawed procedure. Boar semen dilute with freeze extender supplement without or with 20 mg/ml taurine and 20 mg/ml L-glutamine were 51.3%, 51.3% and 52.5%, respectively. There was no significant difference among groups. When straw placed in -20? cooling room at the time intervals of 0, 5, 15 min, then put the straw above LN vapor for another 20, 15 and 5 min, the sperm motility was no significant difference. However, there was no survive if the straw placed into LN directly after storage at -20?for 25 min. When boar semen diluted with MIII (Kiev), Androhep, BTS, Modena before or after frozen- thawed procedure and the result was showed that the sperm motility was lowest at Modena group for fresh semen extender, however, the sperm motility was highest at Modena group for thawed extender. The result indicated that the higher thawed-sperm motility have higher conception rate after AI. Keywords: Boar , Cryopreservation , Motile spermatozoa , Semen , Extender , Trypan blue , Kiev , Modena