冷冻和解冻后经流式细胞术分选的公羊精子功能能力的体外和体内评估
In vitro and in vivo assessment of functional capacity of flow cytometrically sorted ram spermatozoa after freezing and thawing.
作者信息
Hollinshead F K, Gillan L, O'Brien J K, Evans G, Maxwell W M C
机构信息
Centre for advanced Technologies in Animal Genetics and Reproduction, Faculty of Veterinary Science, The University of Sydney, NSW, Australia.
出版信息
Reprod Fertil Dev. 2003;15(6):351-9. doi: 10.1071/RD03060.
The effect of sex sorting and freeze-thawing on the viability and fertility of ram spermatozoa was investigated in the present study. Non-sorted (control) frozen-thawed spermatozoa had a higher motility and forwards progressive motility (FPM) than sorted frozen-thawed spermatozoa (60.9 +/- 2.9% v. 57.0 +/- 3.3% and 4.0 +/- 0.1 v. 3.5 +/- 0.1 FPM, respectively; P < 0.001) after incubation (6 h at 37 degrees C). Sorted and non-sorted (control) frozen-thawed spermatozoa had similar acrosome integrity (73.7 +/- 1.8% v. 75.2 +/- 2.1%, respectively) after thawing and incubation. A greater proportion of sorted spermatozoa displayed chlortetracycline staining patterns that were characteristic of capacitation (22.0 +/- 2.8%; P < 0.05) than non-sorted (control) spermatozoa (15.4 +/- 2.6% B pattern) before freezing. Overall, more sorted frozen-thawed spermatozoa showed patterns characteristic of being acrosome reacted (12.8 +/- 0.7%; P < 0.01) and less were uncapacitated (35.5 +/- 0.6%; P < 0.05) than non-sorted (control) frozen-thawed spermatozoa (7.7 +/- 0.8%; and 38.6 +/- 0.6% for AR and F pattern, respectively). Similar numbers of non-sorted (control) and sorted frozen-thawed spermatozoa migrated through artificial cervical mucus after 1 h (76.4 +/- 11.9 v. 73.9 +/- 11.9 spermatozoa, respectively). The distance travelled by the vanguard spermatozoon was also similar (56.9 +/- 7.8 v. 38.6 +/- 5.8 mm for control and sorted spermatozoa, respectively). Sorted and control frozen-thawed spermatozoa displayed a similar pattern of binding to, and release from, an oviduct epithelial cell monolayer (OECM), but sorted frozen-thawed spermatozoa were released more rapidly (P < 0.05) than non-sorted (control) frozen-thawed spermatozoa. The pregnancy rate was higher for ewes inseminated with 100 x 10(6) (commercial control) frozen-thawed spermatozoa (59%) than for 5, 10, 20 and 40 x 10(6) total sorted frozen-thawed spermatozoa (41% overall; P < 0.001). Insemination of 16 x 10(6) resulted in a higher pregnancy rate (31%) than 10(6) (17%; P < 0.05), but was similar to ewes that received 4 x 10(6) sorted frozen-thawed spermatozoa (24%). Time of insemination (54, 58 and 62 h after sponge removal) had no effect on pregnancy rate. Pregnancy in gonadotrophin-releasing hormone-treated ewes was affected by insemination dose (P < 0.05) but not sperm type (sorted and non-sorted) or ram. Pregnancy was higher after insemination of 40 x 10(6) than 5 or 20 x 10(6) non-sorted (control) or sorted frozen-thawed spermatozoa (70%, 33% and 35%, respectively; P < 0.05). Sorted frozen-thawed spermatozoa may have a shorter viability within the female tract than non-sorted frozen-thawed spermatozoa.
本研究调查了性别分选和冻融对公羊精子活力和受精能力的影响。未分选（对照）的冻融精子在孵育（37℃，6小时）后，其活力和前向运动性（FPM）高于分选的冻融精子（分别为60.9±2.9%对57.0±3.3%，以及4.0±0.1对3.5±0.1 FPM；P<0.001）。分选和未分选（对照）的冻融精子在解冻和孵育后，顶体完整性相似（分别为73.7±1.8%对75.2±2.1%）。与冷冻前未分选（对照）的精子（15.4±2.6% B型）相比，更大比例的分选精子呈现出获能特征性的金霉素染色模式（22.0±2.8%；P<0.05）。总体而言，与未分选（对照）的冻融精子相比，更多分选的冻融精子呈现出顶体反应的特征模式（12.8±0.7%；P<0.01），而未获能的精子较少（35.5±0.6%；P<0.05）（顶体反应和F型分别为7.7±0.8%和38.6±0.6%）。1小时后，未分选（对照）和分选的冻融精子穿过人工宫颈黏液的数量相似（分别为76.4±11.9对73.9±11.9个精子）。先锋精子游动的距离也相似（对照和分选精子分别为56.9±7.8对38.6±5.8毫米）。分选和对照的冻融精子与输卵管上皮细胞单层（OECM）的结合和释放模式相似，但分选的冻融精子比未分选（对照）的冻融精子释放得更快（P<0.05）。用100×10⁶（商业对照）冻融精子输精的母羊妊娠率（59%）高于用5、10、20和40×10⁶个总分选冻融精子输精的母羊（总体为41%；P<0.001）。输精16×10⁶个精子的妊娠率（31%）高于输精10⁶个精子的妊娠率（17%；P<0.05），但与接受4×10⁶个分选冻融精子的母羊相似（24%）。输精时间（去除海绵栓后54、58和62小时）对妊娠率没有影响。促性腺激素释放激素处理的母羊的妊娠受输精剂量影响（P<0.05），但不受精子类型（分选和未分选）或公羊影响。输精40×10⁶个精子后的妊娠率高于输精5或20×10⁶个未分选（对照）或分选冻融精子后的妊娠率（分别为70%、33%和35%；P<0.05）。分选的冻融精子在雌性生殖道内的存活时间可能比未分选的冻融精子短。
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