Bathgate R, Mace N, Heasman K, Evans G, Maxwell W M C, de Graaf S P
Faculty of Veterinary Science, The University of Sydney, Sydney, NSW, Australia.
Reprod Domest Anim. 2013 Dec;48(6):893-8. doi: 10.1111/rda.12182. Epub 2013 May 23.
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(®) ) 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) 0.25 ml pellets on dry ice or 0.25 ml 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.
山羊精子的成功性别分选以及随后预分选后代的出生尚未见报道。因此，开展了一系列实验以制定精子分选（使用改良的流式细胞仪MoFlo SX(®)）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）分选后收集到萨拉蒙氏或Tris收集培养基中，（ii）在添加5%、10%或20%蛋黄的Tris - 柠檬酸盐 - 葡萄糖培养基中冷冻，（iii）在干冰上制成0.25 ml颗粒或在程序降温冷冻仪中制成0.25 ml细管。分选后和解冻后的精子质量通过活力（计算机辅助精子分析）、存活率和顶体完整性（碘化丙啶/异硫氰酸荧光素 - 花生凝集素）进行评估。以颗粒形式冷冻的性别分选山羊精子解冻后的活力和存活率显著高于以细管形式冷冻的精子。收集培养基和不同的蛋黄浓度对所测试的精子参数没有影响。然后分别通过异源卵子结合试验和对萨能山羊母羊进行子宫内人工授精，测试了用这种最佳方案生产的性别分选山羊精子的体外和体内受精能力。性别分选的山羊精子与绵羊卵子透明带结合的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）。用性别分选的精子进行子宫内人工授精后，38%（5/13）的母羊产仔，其中83%（3/5）的后代为预期性别。用未分选的精子进行人工授精的母羊产仔率为50%（3/6），性别比例为3∶1（雌性∶雄性）。本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分选后代。
