title: Effects of Hoechst33342 staining on the viability and flow cytometric sex-sorting of frozen-thawed ram sperm.
abstract: 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 μM, 120 μM, 160 μM, 200 μM, 240 μM, or 320 μM) for 45 min to evaluate effects of dye dose. The frozen-thawed sperm were stained with 160 μM 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 μM. 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 160μM 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 μM 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.
authors.affiliation: Yunnan Animal Science and Veterinary Institute, Jindian, Panlong County, Kunming City, Yunnan Province, China; Yunnan Provincial Meat Caprine Engineering Research Center, Jindian, Panlong County, Kunming City, Yunnan Province, China; Yunnan Provincial Engineering Laboratory of Animal Genetic Resource Conservation and Germplasm Enhancement, Jindian, Panlong County, Kunming City, Yunnan Province, China.
authors.forename: Guo Bo
authors.initials: GB
authors.lastname: Quan
authors.affiliation: Yunnan Animal Science and Veterinary Institute, Jindian, Panlong County, Kunming City, Yunnan Province, China; Yunnan Provincial Meat Caprine Engineering Research Center, Jindian, Panlong County, Kunming City, Yunnan Province, China; Yunnan Provincial Engineering Laboratory of Animal Genetic Resource Conservation and Germplasm Enhancement, Jindian, Panlong County, Kunming City, Yunnan Province, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Kunming Institute of Zoology, Chinese Academy of Sciences (CAS), Wuhua County, Kunming City, Yunnan Province, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.affiliation: Yunnan Animal Science and Veterinary Institute, Jindian, Panlong County, Kunming City, Yunnan Province, China; Yunnan Provincial Meat Caprine Engineering Research Center, Jindian, Panlong County, Kunming City, Yunnan Province, China; Yunnan Provincial Engineering Laboratory of Animal Genetic Resource Conservation and Germplasm Enhancement, Jindian, Panlong County, Kunming City, Yunnan Province, China.
authors.forename: Guo Quan
authors.initials: GQ
authors.lastname: Wu
authors.affiliation: Yunnan Animal Science and Veterinary Institute, Jindian, Panlong County, Kunming City, Yunnan Province, China; Yunnan Provincial Meat Caprine Engineering Research Center, Jindian, Panlong County, Kunming City, Yunnan Province, China; Yunnan Provincial Engineering Laboratory of Animal Genetic Resource Conservation and Germplasm Enhancement, Jindian, Panlong County, Kunming City, Yunnan Province, China.
authors.forename: Dong Jiang
authors.initials: DJ
authors.lastname: Li
authors.affiliation: Yunnan Animal Science and Veterinary Institute, Jindian, Panlong County, Kunming City, Yunnan Province, China; Yunnan Provincial Meat Caprine Engineering Research Center, Jindian, Panlong County, Kunming City, Yunnan Province, China; Yunnan Provincial Engineering Laboratory of Animal Genetic Resource Conservation and Germplasm Enhancement, Jindian, Panlong County, Kunming City, Yunnan Province, China.
authors.forename: Yi Na
authors.initials: YN
authors.lastname: Ni
authors.affiliation: Yunnan Animal Science and Veterinary Institute, Jindian, Panlong County, Kunming City, Yunnan Province, China; Yunnan Provincial Meat Caprine Engineering Research Center, Jindian, Panlong County, Kunming City, Yunnan Province, China; Yunnan Provincial Engineering Laboratory of Animal Genetic Resource Conservation and Germplasm Enhancement, Jindian, Panlong County, Kunming City, Yunnan Province, China.
authors.forename: Chun Rong
authors.initials: CR
authors.lastname: Lv
authors.affiliation: Yunnan Animal Science and Veterinary Institute, Jindian, Panlong County, Kunming City, Yunnan Province, China; Yunnan Provincial Meat Caprine Engineering Research Center, Jindian, Panlong County, Kunming City, Yunnan Province, China; Yunnan Provincial Engineering Laboratory of Animal Genetic Resource Conservation and Germplasm Enhancement, Jindian, Panlong County, Kunming City, Yunnan Province, China.
authors.forename: Lan
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Yunnan Animal Science and Veterinary Institute, Jindian, Panlong County, Kunming City, Yunnan Province, China; Yunnan Provincial Meat Caprine Engineering Research Center, Jindian, Panlong County, Kunming City, Yunnan Province, China; Yunnan Provincial Engineering Laboratory of Animal Genetic Resource Conservation and Germplasm Enhancement, Jindian, Panlong County, Kunming City, Yunnan Province, China. Electronic address: qionghuahong@163.com.
authors.forename: Qiong Hua
authors.initials: QH
authors.lastname: Hong
mesh_terms: D000177:Acrosome / Q000187:drug effects*; D000818:Animals; D001562:Benzimidazoles / Q000737:chemistry / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D005307:Fertilization in Vitro; D005434:Flow Cytometry / Q000379:methods; D005615:Freezing; D008297:Male; D010718:Phosphatidylserines / Q000378:metabolism; D037121:Plant Lectins / Q000737:chemistry; D012661:Semen / Q000187:drug effects; D012662:Semen Preservation / Q000379:methods*; D012756:Sheep; D013081:Sperm Motility / Q000187:drug effects*; D013094:Spermatozoa / Q000187:drug effects; D013194:Staining and Labeling
chemical_list: D001562:Benzimidazoles; D010718:Phosphatidylserines; D037121:Plant Lectins; C036292:pea lectin; C017807:bisbenzimide ethoxide trihydrochloride
keywords: Cytometric sorting; Freezing; Hoechst33342; Ram sperm; Thawing