- 期刊
冷凍保護劑與平衡時間對微滴玻璃化冷凍之山羊早期胚後續發育之影響
Developmental competence of early caprine embryos vitrified with various cryoprotectant formulae and equilibrium time
摘要
本試驗目的是利用微滴玻璃化冷凍（micro-drop vitrification）技術進行山羊早期胚冷凍保存之研究，增加冷凍早期胚解凍後之存活與後續發育之能力，以利將來人工生殖相關技術研發及種原保存工作。經產母羊經超級排卵（superovulation）處理及配種後之第2－3天，以外科手術自輸卵管回收早期胚進行玻璃化冷凍。試驗一以濃度16.5%EG + 16.5% DMSO冷凍保護劑進行不同發育階段早期胚微滴玻璃化冷凍。試驗二比較不同濃度冷凍保護劑進行8細胞期胚之玻璃化冷凍。試驗三評估不同冷凍保護劑配方進行4細胞期胚之玻璃化冷凍。試驗四探討第一階段不同平衡時間繼之以20.0% EG + 20.0% DMSO冷凍保護劑進行4細胞期胚之玻璃化冷凍效果。試驗一之結果顯示，4細胞期之山羊胚於解凍後無法繼續發育，而8細胞期之山羊胚於解凍後發育至桑椹胚者為22.2%，16-細胞期之山羊胚於解凍後發育至桑椹胚者為50.0%，而各細胞期之山羊胚均未發育至囊胚階段。於試驗二，利用16.5%EG + 16.5% DMSO進行8細胞期山羊胚之玻璃化冷凍再解凍後，發育至桑椹胚為9.1%，但未發育至囊胚；利用20.0%EG + 20.0% DMSO進行8細胞期山羊胚之玻璃化冷凍再解凍後，發育至桑椹胚為20.0%，而囊胚率為6.7%。於試驗三，利用20.0%EG + 20.0% DMSO進行早期胚之玻璃化冷凍再解凍後，發育至8細胞期為26.1%，而囊胚率為4.3%；利用25.0%EG + 25.0% glycerol進行早期胚之玻璃化冷凍再解凍後，發育至8細胞期為11.1%，並未發育至囊胚。於試驗四，利用20.0%EG + 20.0% DMSO進行早期胚之玻璃化冷凍前，於低濃度冷凍保護劑（10.0% EG + 10.0% DMSO）下平衡時間45秒再解凍後，發育至8細胞期為25.0%，而無囊胚發育；平衡時間若縮減為35秒，其再解凍後發育至8細胞期者為33.3%，而囊胚率為12.8%。證實預平衡時間為35秒且繼之以20.0% EG + 20.0% DMSO冷凍保護液進行山羊早期胚微滴玻璃化冷凍保存，可成功發育至囊胚期。
並列摘要
The objective of this study is to examine the effects of different cryoprotectant formulas on the development of post-thawed caprine embryos vitrified at early developmental stage. Early stages of embryos were collected from the oviducts of superovulated does by surgical method on days 2-3 after natural mating. In Experiment 1, 4-, 8- and 16-cell stage embryos were vitrified in solution containing 16.5% EG + 16.5% DMSO. In Experiment 2, 8-cell stage embryos were vitrified in solution containing 16.5% EG + 16.5% DMSO or 20.0% EG + 20.0% DMSO. In Experiment 3, 4-cell stage embryos were vitrified in solution with 20.0% EG + 20.0% DMSO or 25.0% EG + 25.0% DMSO. In Experiment 4, 4-cell stage embryos were equilibrated with various period time before vitrification in 20.0% EG + 20.0% DMSO. The morula rates of vitrified-thawed embryos at 4, 8 and 16-cell stages in Experiment 1 were 0, 22.0 and 50.0%, respectively. No embryos developed to the blastocyst stage. In Experiment 2, the morula and blastocyst rates of 8-celled embryos vitrified in 16.5% EG + 16.5% DMSO and 20.0% EG + 20.0% DMSO were 9.1% and 0% and 20.0% and 6.7%, respectively. In Experiment 3, the cleavage and blastocyst rates of 4-celled embryos vitrified in the solution containing 20.0% EG + 20.0% DMSO and 25.0% EG + 25.0% glycerol were 26.1% and 4.3%, 11.1% and 0%, respectively. In Experiment 4, the cleavage and blastocyst rates of 4-celled embryos balanced for 45 and 35 seconds prior to vitrification in the solution containing 20.0% EG + 20.0% DMSO were 25% and 0%, 33.3% and 12.8%, respectively. These results indicate that early stage embryos are able to successfully develop to the blastocyst stage after vitrification in the solution containing 20.0% EG + 20.0% DMSO and equilibrating for 35 seconds prior to vitrification.
延伸閱讀
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