Keskintepe L, Simplicio A A, Brackett B G
Department of Physiology & Pharmacology, College of Veterinary Medicine, University of Georgia, Athens 30602, USA.
Theriogenology. 1998 May;49(7):1265-74. doi: 10.1016/S0093-691X(98)00074-0.
The feasibility of using frozen-thawed semen in caprine IVF outside the breeding season was investigated. Electroejaculated spermatozoa from a Nubian buck were washed twice and then frozen in skim milk- or in egg yolk-based extenders. Goat oocytes were matured and inseminated by frozen-thawed spermatozoa selected by swim-up. In vitro fertilization was performed in a modified defined medium (mDM), altered experimentally, for 24 h. Embryos were cultured in 50 microL of c-SOF + NEA for 9 d. The percentages of oocytes exposed to heparin-capacitated spermatozoa, (previously cryopreserved in skim milk-based extender) that cleaved, reached morula, blastocyst and expanded blastocyst stages were 82.8, 57.1, 35.7 and 30.0%, respectively. Without heparin treatment the rates for cleavage, morula, blastocyst and expanded blastocyst stages were 44.3, 31.4, 18.6 and 8.6%, respectively. Therefore, heparin treatment was included in sperm capacitation. Use of spermatozoa with BSA in the IVF medium yielded no cleavage. Although extenders containing 8 to 20% egg yolk enabled good sperm motility after cryopreservation, in vitro fertilizing ability was compromised under our conditions. By contrast, semen commercially processed in season in an egg yolk-based diluent remained effective for IVF. The highest proportion of blastocysts resulted from the use of spermatozoa diluted in a skim milk extender, heparin capacitation, and insemination in medium containing lamb serum.
研究了在繁殖季节外使用冻融精液进行山羊体外受精的可行性。对一只努比亚公羊电刺激采得的精子洗涤两次，然后分别在基于脱脂乳或蛋黄的稀释液中冷冻。山羊卵母细胞经成熟培养后，用通过上浮法筛选出的冻融精子进行授精。体外受精在经实验改变的改良限定培养基（mDM）中进行24小时。胚胎在50微升的c-SOF + NEA中培养9天。暴露于肝素诱导获能精子（先前保存在基于脱脂乳的稀释液中）的卵母细胞，其分裂、达到桑葚胚、囊胚和扩张囊胚阶段的百分比分别为82.8%、57.1%、35.7%和30.0%。未经肝素处理时，分裂、桑葚胚、囊胚和扩张囊胚阶段的比率分别为44.3%、31.4%、18.6%和8.6%。因此，精子获能过程中加入了肝素处理。在体外受精培养基中使用含牛血清白蛋白的精子未产生分裂现象。尽管含有8%至20%蛋黄的稀释液能使精子在冷冻保存后保持良好的活力，但在我们的条件下，其体外受精能力受到影响。相比之下，在繁殖季节用基于蛋黄的稀释液进行商业处理的精液对体外受精仍然有效。使用在脱脂乳稀释液中稀释的精子、肝素诱导获能并在含羔羊血清的培养基中授精时，产生的囊胚比例最高。
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