- 期刊
稀釋與冷凍處理過程對山羊冷凍精液解凍後存活率與活力之影響
Effects of Dilution and Freezing Process on Survival Rate and Motility in Frozen-Thawed Goat Semen
摘要
本試驗之目的在探討各種可能對山羊精液冷凍再解凍後精子存活率及活力之影響因子，以期發展出適合山羊精液冷凍保存條件，作為未來生產推廣乳羊冷凍精液及人工授精之用。山羊精液以假陰道採取後，立即以精子分析儀（Hamilton-Thorn HTM-C型）進行精子濃度、存活率、活力等精液性狀之評估，並經適當稀釋至最終濃度為每支麥管含1.5×10^8個精子，在精液稀釋冷凍過程中比較下列不同處理，以了解其對解凍後精液性狀之影響：（1）稀釋前先行離心去除精漿與否（2）第一段稀釋液為含10%脫脂乳粉或20%蛋黃－枸椽酸鈉之稀釋液（3）第二段稀釋液含3.5%或7.0%甘油之冷凍保護劑（4）以0.25 ml或0.5 ml之麥管填充後冷凍（5）以二步驟降溫法或單步驟降溫法進行冷凍。試驗結果顯示，（A）使用脫脂乳粉為第一段稀釋液者，解凍後之精液性狀極顯著優於以蛋黃－枸椽酸鈉稀釋者（P＜0.01）（存活率分別為57.5±11.9% vs. 17.5±10.1%；活力則分別為2.9±0.5 vs. 1.3±0.5）。（B）採精後是否先行離心去除精漿對於以脫脂乳粉處理者之解凍後精液性狀之影響不顯著。（C）比較3.5%或7.0%甘油為冷凍保護劑，不論精漿存在與否，其對於解凍後之存活率並無顯著之影響；然在活力方面，未離心去除精漿並以7.0%甘油者，解凍後之活力顯著優於以3.5%甘油者（P＜0.05）（活力分別為3.1±0.5 vs. 2.4±0.3）。（D）以二步驟降溫法進行冷凍降溫，解凍後之精液性狀極顯著優於單步驟法者（P＜0.01）（存活率與活力分別為58.5±12.1% vs. 22.9±11.9%；3.0±0.5 vs. 1.6±0.4）。（E）以0.25 ml或0.5 ml麥管填充並配合二步驟降溫法進行冷凍者，解凍後之精液性狀並無顯著之差異。冷凍精液之受精能力以人工授精方式評估，獲得兩母山羊群之受胎率分別為36.8%與30.6%，顯示以此方法保存之冷凍精液在解凍之後仍具有受精能力。本試驗結果顯示使用10%脫脂乳粉為稀釋液，山羊新鮮精液不必離心去除精漿，其結果並不影響解凍後精液性狀，且在稀釋液中含7.0%甘油作為冷凍保護劑，及採用0.25 ml或0.5 ml麥管填充，並以二步驟降溫法進行冷凍者，可得到最佳之冷凍效果，且解凍後仍具有良好之受精能力，此可做為未來山羊精液冷凍保存及應用推廣之參考。
並列摘要
The aim of this study was to evaluate the effects of several factors during dilution and freezing procedure on the survival rate and the motility of spermatozoa in frozen-thawed goat semen. Semen was collected from Alpine and Saanen bucks by using artificial vagina. Concentration and motility of sperm, and percentage of live sperm were measured by sperm analyzer (Hamilton-Thorn HTM-C model). Semen were finally diluted to contain 1.5 × 10^8 spermatozoa/straw. Factors affecting the qualities of frozen-thawed semen were examined as follows: (1) keeping semen intact or removal of seminal plasma by centrifugation before dilution. (2) using 10% of skim milk or 20% of egg yolk-sodium citrate in the first diluent. (3) using 3.5% or 7.0% of glycerol in the final dilution. (4) loading into 0.25 ml or 0.5 ml of straw for freezing. (5) freezing stepwise before the straw was plunged into liquid nitrogen: -80 C for 2 minutes then -110 C for 3 minutes (two steps) or -80 C for 10 minutes (one step). The results of this experiment were: (A) the survival rate and motility of sperm in the frozen-thawed semen diluted with skim milk were significantly higher than those diluted with egg yolk-sodium citrate diluent irrespective of existence or removal of seminal plasma before dilution (P＜0.01) (57.5 ± 11.9% vs. 17.5 ± 10.1%；2.9 ± 0.5 vs. 1.3 ± 0.5, respectively). (B) existence of seminal plasma did not affect the survival rate and motility of sperm in skim milk diluted semen both in prefreezing and post thawing stages. (C) although the survival rate of semen was not affected by the concentration of glycerol, the motility of sperm in the group of 7.0% glycerol was significantly higher than that in 3.5% glycerol group (P＜0.05) (3.1 ± 0.5 vs. 2.4 ± 0.3, respectively.) (D) survival rate and motility of semen frozen by two steps were significantly higher than the one step did (P＜0.01) (58.5 ± 12.1% vs. 22.9 ± 11.9%； 3.0 ± 0.5 vs. 1.6 ± 0.4, respectively). (E) no significant differences in the survival rate or the motility were observed between groups with different loading volumes of straw. The fertility of frozen-thawed semen was proven good by artificially inseminating two herds of doe with a result of 36.8% and 30.6% conception rate. These results demonstrated that goat semen together with seminal plasma diluted with 10% of skim milk containing 7.0% of glycerol, then loaded in 0.25 ml or 0.5 ml of straw, and frozen by two freezing steps were the optimal treatments for freezing goat semen.