拉布拉多犬X/Y精子分选及性别控制冷冻精液制备实验研究

Study on Cryopreservation of Flow-cytometrically Sorted X- or Y- chromosome Bearing Sperm on Labrador Retriever

目的探讨流式细胞分选技术在分离拉布拉多犬X、Y精子方面的应用，并研究拉布拉多犬性别控制（性控）冷冻精液的制备方法。方法人工按摩刺激法采集8只拉布拉多种公犬精液，利用SX_MOFLO流式细胞仪展开X、Y精子分离实验，并对分离后的X／Y精子冷冻、解冻后进行精液重分析和品质鉴定。结果流式细胞仪分选的X／Y精子纯度达到89％以上（89％～91％），X精子平均分离速度（4260个／s）极显著高于Y精子分离速度（3930个／s）（P〈0．01）；常规冷冻精液和X、Y型冷冻精液性别比差异显著（P〈0．05）；冻融后活率则无明显差异（P〉0．05）；常规冷冻精液与X、Y型冷冻精液解冻后1h存活指数差异明显（P〈0．05），分别为0．251±0．006、0．054±0．007和0．048±0．006；冻融后顶体完整率差异显著（P〈0．05），分别为77．00％±0．98％、84．66％±1．22％和84．01％±1．23％。结论用流式细胞分选技术可以获得高纯度的拉布拉多犬X／Y冷冻精液，性别比和精液品质达到低剂量人工授精的要求。

Objective To investigate the application of flow cytometric technology in separating X or Y sperm from Labrador retriever, and to define a protocol for producing sex-sorted frozen semen. Method The semen were collected from 8 Labrador retrievers by manual stimulation and transported in one hour, and then the semen were separated into X- and Y- chromosome by SX MOFLO flow cytometric technology, a modified high-speed cell sorter, and finally frozen thawed. Sorted X- or Y- sperm and unsorted sperm as control were reanalyzed and quality assessed. Results The purities of flow sorted sex chromosomes in spermatozoa of three dogs ranged from 89% to 91%. The average separation velocity showed significantly differences between sorted X- sperm （4 260/s） and Y- sperm （3 930/s）. The sex ratio showed significantly differences between frozen- thawed sorted X- or Y- chro- mosome bearing sperm （89.11%, 90.67%） and the control. The forward progressive motility was not significantly different between sorted sperm and the control. The survive index on sorted sperm was significantly lower （0.251± 0.006／0.054 ± 0.007, P〈0.05） than the control （0.048 ±0.006）. But the post thaw acrosome integrity on sorted sperm was significantly higher （84.66 ± 1.22／84.01 ±1.23, P〈0.05） than the control （77.00 ±0.98）. Conclusion It was possible to obtain sorted frozen- thawed Labrador retriever sperm for AI by using flow cytometry techniques.