稀釋液組成分與保存方法對臺灣梅花鹿精液品質之影響

外文標題: 
Effects of the Components of Semen Extenders and Storage Methods on the Semen Quality in the Formosan Sika Deer Stags
校院系所: 
屏東科技大學畜產系
指導教授: 
劉炳燦
出版年份: 
2007年
主題類別: 
摘要: 

臺灣梅花鹿(Formosan Sika deer; Cervus nippon taiouanus)為鹿科(Cervidae)動物之臺灣特有亞種,具有短日照季節性配種(short day seasonal breeding)之特性,本文之目的在探討利用不同稀釋液搭配不同冷凍方法,以及經解膠處理與否對臺灣梅花鹿新鮮精液及冷凍解凍後精液品質之影響。精液均利用電激採精方法採得,其活力≧4級以上者方供後續之試驗用,新鮮精液分別以Tris based(T組)、Glycine based (G組)和Tris+ Citric acid based (TC組)3種稀釋液稀釋後,於4℃冷藏保存(研究一),再分別經慢速冷凍(slow freezing)、不含抗凍保護劑之玻璃化法(cryoprotectant-free vitrification)予以冷凍保存(研究二),亦探討不同解膠(degelatification)方法(研究三),對鹿隻精子活力(sperm motility score, SMS)、精子存活率(sperm viability)、精子頭帽完整性(sperm acrosomal integrity)及精子穿透能力(sperm penetration ability)等精液品質相關性狀之影響。結果顯示,臺灣梅花鹿之精液以G組或TC組稀釋液進行4℃冷藏保存7天,仍可保持其相較於第0天之精子活力57%、存活率81%、頭帽完整性83%及pH值降低5%,冷藏保存第7天之精子,經獲能處理後對ZFO倉鼠卵穿透率與精子穿卵數於G組和TC組間皆無顯著差異(約為97% 和38個精子),惟TC組活力、存活率、頭帽完整性及穿透率皆有較佳之趨勢(研究一)。以G組或TC組稀釋液添加冷凍保護劑進行慢速冷凍保存或玻璃化冷凍保存,冷凍解凍後其精子品質相似(各約為SMS: 3.3; viability: 53%; acrosomal integrity: 63%),惟經解凍培養試驗顯示3 h後,玻璃化法之存活率顯著高於慢速冷凍法者(29 vs. 23%, P < 0.05),綜合稀釋液種類與冷凍方法之解凍後培養3 h 之結果,以TC組為稀釋液進行玻璃化法之冷凍保存效果較佳(研究二)。新鮮含膠質精液,添加發情母鹿陰道黏液同時以TC組稀釋液,其精子存活率及頭帽完整性皆較佳於以酵素解膠之其他各組(分別約為69.3 vs. 66% 和73.8 vs. 67%, P < 0.05),於酵素解膠之各組中,則以添加25% Collagenase(1 mg/mL)冷凍解凍後精子活力(2.9級)、存活率(50%)和頭帽完整性(63%)則皆較高(研究三)。綜合本研究之各項結果顯示,臺灣梅花鹿之精液可適用於以Glycine based或Tris + Citric acid based之稀釋液進行7天冷藏保存,或添加冷凍保護劑glycerol以慢速冷凍保存及無添加冷凍保護劑進行玻璃化冷凍保存,亦可利用母鹿陰道黏液或25% Collagenase進行新鮮精液解膠並進行冷凍保存,其品質均可於臺灣梅花鹿之人工授精用。

外文摘要: 

Formosan Sika deer (Cervus nippon taiouanus) is classified to be a Taiwan specific sub-species with the characteristic of short day seasonal breeding. The aim of this study is to estimate the effect of semen extenders, methods of frozen and degelatification on the quality of fresh liquid and frozen-thrawed semen of the Formosan Sika deer stags. Only the semen collected by electro-ejaculation with sperm motility score (SMS) ≧4 was used in the three studies in this thesis. Fresh semen was diluted with 3 kinds extenders including Tris based (T), Glycine based (G) and Tris+ Citric acid based (TC) at room temperature, and cooled to 4℃ for liquid semen storage. The semen quality including SMS, sperm viability, sperm acrosomal integrity and sperm penetration ability were detected during cooling storage (study 1). The sperm quality of frozen-thawed semen by slow freezing or cryoprotectant-free vitrification method was evaluated (study 2), and the effect of degelatification methods on semen quality was estimated (study 3). Result showed that the fresh semen stored for 7 days at 4℃ (liquid semen) with extenders G and TC, the sperm motility, viability and acrosomal integrity decreased to 57%, 81% and 83% compared to that in 0 day but the pH value was decreased to 5%. The sperm penetration ability, including penetration ratio (PR) and sperm penetration per oocyte (SPO) detected with hamster ZFO of G and TC extenders were not significantly different (about 97% and 38 sperm), but the SMS, viability, acrosomal integrity and penetration ratio (PR) were slightly higher in TC than G extender (study 1). The quality of slow freezing and vitrification frozen-thawed semen were the same (estimated SMS: 3.3; viability: 53%; acrosomal integrity: 63%). But the sperm viability of vitrification were significant higher than slow freezing (29 vs. 23%, P < 0.05) after 3 h incubation. Overall the effect of the kinds of extenders and frozen methods, the TC extender was suitable for dilution and vitrificated cryopreservation of the semen (study 2). The sperm viability and acrosomal integrity of gelated semen degelatificated with vaginal mucus and in TC extender was significantly higher than with enzyme (estimated 69.3 vs. 66% and 73.8 vs. 67%, P < 0.05). The sperm motility score (2.9), viability (50%) and acrosomal integrity (63%) was obtained by 25% collagenase degelatification was the highest among the other enzymes used (study 3). To conclude the results in this thesis, the Glycine based or Tris + Citric acid based extender could be used in 4℃ storage for 7 days as liquid semen. The gelated semen that degelatificated with vaginal mucus or 25% collagenase degelatificated or the non-gelated semen could be stored by slow freezing or cryoprotectant-free vitrification. The semen quality obtained in this thesis could be suitable for artificial insemination.