Birth of Kids After Artificial Insemination with Sex-Sorted, Frozen-Thawed Goat Spermatozoa
Birth of Kids After Artificial Insemination with Sex-Sorted, Frozen-Thawed Goat Spermatozoa
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DOI：
10.1111/rda.12182
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发表时间：
2013-12-01
影响因子：
1.7
通讯作者：
de Graaf, S. P.
中科院分区：
文献类型：
--
作者：
Bathgate, R.;Mace, N.;de Graaf, S. P.
内容 山羊精子的成功性别分选以及随后出生的预性别儿童尚未有报道。因此，进行了一系列实验来开发精子分选（使用改进的流式细胞仪 MoFlo SX (R)）和山羊精子冷冻保存的方案。将萨能山羊精子（n = 2 只雄性）（i）分选后收集到 Salamon's 或 Tris catch 培养基中，（ii）冷冻在补充有 5%、10% 或 20% 蛋黄的 Tris-柠檬酸盐-葡萄糖培养基中（iii）干冰上的 0.25ml 颗粒或控速冰箱中的 0.25ml 吸管。通过活力（CASA）、活力和顶体完整性（PI/FITC-PNA）评估分选后和解冻后的精子质量。与冷冻在吸管中的精子相比，冷冻在颗粒中的性别分选山羊精子在解冻后表现出显着更高的活力和活力。捕获介质和不同的蛋黄浓度对测试的精子参数没有影响。然后分别通过异源卵子结合测定和萨能山羊宫内人工授精的方式测试用该最佳方案产生的性别分选山羊精子的体外和体内生育能力。性别分选的山羊精子与绵羊卵透明带的结合数量与未分选的山羊精子、未分选的公羊精子和性别分选的公羊精子相似（p>0.05）。使用按性别分类的精子进行宫内人工授精后，38% (5/13) 的受孕者生下的孩子中，83% (3/5) 的孩子与预期性别相同。使用未分选的精子进行授精是否达到了 50% (3/6) 的受精率和 3:1 (F:M) 的性别比。这项研究首次证明，山羊精子可以通过流式细胞术进行性别分选，成功冷冻并用于生产预性别的孩子。
Contents Successful sex-sorting of goat spermatozoa and subsequent birth of pre-sexed kids have yet to be reported. As such, a series of experiments were conducted to develop protocols for sperm-sorting (using a modified flow cytometer, MoFlo SX (R)) and cryopreservation of goat spermatozoa. Saanen goat spermatozoa (n=2 males) were (i) collected into Salamon's or Tris catch media post-sorting and (ii) frozen in Tris-citrate-glucose media supplemented with 5, 10 or 20% egg yolk in (iii) 0.25ml pellets on dry ice or 0.25ml straws in a controlled-rate freezer. Post-sort and post-thaw sperm quality were assessed by motility (CASA), viability and acrosome integrity (PI/FITC-PNA). Sex-sorted goat spermatozoa frozen in pellets displayed significantly higher post-thaw motility and viability than spermatozoa frozen in straws. Catch media and differing egg yolk concentration had no effect on the sperm parameters tested. The in vitro and in vivo fertility of sex-sorted goat spermatozoa produced with this optimum protocol were then tested by means of a heterologous ova binding assay and intrauterine artificial insemination of Saanen goat does, respectively. Sex-sorted goat spermatozoa bound to sheep ova zona pellucidae in similar numbers (p>0.05) to non-sorted goat spermatozoa, non-sorted ram spermatozoa and sex-sorted ram spermatozoa. Following intrauterine artificial insemination with sex-sorted spermatozoa, 38% (5/13) of does kidded with 83% (3/5) of kids being of the expected sex. Does inseminated with non-sorted spermatozoa achieved a 50% (3/6) kidding rate and a sex ratio of 3:1 (F:M). This study demonstrates for the first time that goat spermatozoa can be sex-sorted by flow cytometry, successfully frozen and used to produce pre-sexed kids.