Effects of Hoechst33342 staining on the viability and flow cytometric sex-sorting of frozen-thawed ram sperm

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	Effects of Hoechst33342 staining on the viability and flow cytometric sex-sorting of frozen-thawed ram sperm
	Quan GB 1,2,3; Ma Y 1,2,3; Li J 4; Wu GQ 1,2,3; Li DJ 1,2,3; Ni YN 1,2,3; Lv CR 1,2,3; Zhu L 1,2,3; Hong QH[*]1,2,3
刊名	CRYOBIOLOGY
	2015
卷号	70 期号:1 页码:23-31
关键词	Ram sperm Freezing Thawing Hoechst33342 Cytometric sorting
通讯作者	gionghuahong@163.com
合作状况	其它
英文摘要	Cytometric sorting of frozen-thawed sperm can overcome difficulties caused by the unavailability of sorting facilities on farms where semen is collected from male livestock. In order to optimize the cytometric sex-sorting procedure, effects of Hoechst33342 staining on the viability and cytometric sorting efficiency of frozen-thawed ram sperm were evaluated. The frozen-thawed sperm were stained with Hoechst33342 at various dye concentrations (80 mu M, 120 mu M, 160 mu M, 200 mu M, 240 mu M, or 320 mu M) for 45 min to evaluate effects of dye dose. The frozen-thawed sperm were stained with 160 mu M Hoechst33342 for various durations (0 min, 15 min, 30 min, 45 min, 60 min, 75 min, or 90 min) to evaluate effects of staining duration. Sperm motility and moving velocity were analyzed using a computer-assisted sperm analysis system (CASAS). Acrosome status, membrane integrity, and distribution of phosphatidylserine (PS) in Hoechst33342-stained sperm were analyzed using flow cytometry after staining with fluorescein isothiocyanate-labeled lectin from pisum sativum (FITC-PSA), Annexin V, or propidium iodide (PI). The fertility of Hoechst33342-stained sperm was analyzed by in vitro fertilization (IVF). A high-speed cell sorter was used to evaluate effects of Hoechst33342 staining on cytometric sex-sorting of frozen-thawed sperm. The motility, moving velocity, membrane integrity, and PS distribution of Hoechst33342-stained sperm were significantly different from that of immediately thawed sperm (P < 0.05). However, there is no significant difference existing among the Hoechst33342-stained groups with respect to the above evaluated parameters. Additionally, along with the staining durations, the adverse effects of the staining procedure on sperm showed a steady increase. However, Hoechst33342 staining did not damage acrosome and in vitro fertilizing capability of frozen-thawed ram sperm. Results of cytometric sorting indicated that frozen-thawed sperm can be efficiently sorted into two sperm populations with X and Y chromosome when the Hoechst33342 concentration was 160 mu M. Moreover, when the staining duration was equal to or longer than 45 min, the frozen-thawed sperm can be successfully sorted in the presence of 160 mu M Hoechst33342. In conclusion, Hoechst33342 staining can detrimentally influence viability of frozen-thawed ram sperm except acrosome and in vitro fertilizing capability. Accordingly, the minimum values of Hoechst33342 concentration and staining duration can be set at 160 mu M and 45 min respectively. However, the maximum values of Hoechst33342 concentration and staining duration were not determined based on the current study. Further research on how to reduce injuries caused by freezing, thawing, and Hoechst33342 staining on frozen-thawed ram sperm is needed.
收录类别	SCI
资助信息	This investigation was funded by the National Natural Science Foundation of China (Grant No. 31360551), the National Wool Caprine Industrial Technology System (Grant No. CARS-40), and the National Infrastructure of Animal Genetic Resources (NIAGR).
语种	英语
WOS记录号	WOS:000348631900004
公开日期	2015-03-06
内容类型	期刊论文
源URL	[http://159.226.149.42:8088/handle/152453/8272]
专题	管理、支撑部门_大型仪器中心
作者单位	1.Yunnan Animal Science and Veterinary Institute, Jindian, Panlong County, Kunming City, Yunnan Province, China 2.Yunnan Provincial Meat Caprine Engineering Research Center, Jindian, Panlong County, Kunming City, Yunnan Province, China 3.Yunnan Provincial Engineering Laboratory of Animal Genetic Resource Conservation and Germplasm Enhancement, Jindian, Panlong County, Kunming City, Yunnan Province, China 4.Kunming Institute of Zoology, Chinese Academy of Sciences (CAS), Wuhua County, Kunming City, Yunnan Province, China
推荐引用方式 GB/T 7714	Quan GB,Ma Y,Li J,et al. Effects of Hoechst33342 staining on the viability and flow cytometric sex-sorting of frozen-thawed ram sperm[J]. CRYOBIOLOGY,2015,70(1):23-31.
APA	Quan GB.,Ma Y.,Li J.,Wu GQ.,Li DJ.,...&Hong QH[*].(2015).Effects of Hoechst33342 staining on the viability and flow cytometric sex-sorting of frozen-thawed ram sperm.CRYOBIOLOGY,70(1),23-31.
MLA	Quan GB,et al."Effects of Hoechst33342 staining on the viability and flow cytometric sex-sorting of frozen-thawed ram sperm".CRYOBIOLOGY 70.1(2015):23-31.