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

• Published in: Cryobiology 2015-02, Vol.70 (1), p.23-31
• Main Authors: Quan, Guo Bo, Ma, Yuan, Li, Jian, Wu, Guo Quan, Li, Dong Jiang, Ni, Yi Na, Lv, Chun Rong, Zhu, Lan, Hong, Qiong Hua
• Format: Article
• Language: English
• Subjects: Acrosome - drug effects; Animals; Benzimidazoles - chemistry; Benzimidazoles - pharmacology; Cell Membrane - drug effects; Cell Survival; Cryopreservation - methods; Cytometric sorting; Fertilization in Vitro; Flow Cytometry - methods; Freezing; Hoechst33342; Male; Phosphatidylserines - metabolism; Pisum sativum; Plant Lectins - chemistry; Ram sperm; Semen - drug effects; Semen Preservation - methods; Sheep; Sperm Motility - drug effects; Spermatozoa - drug effects; Staining and Labeling; Thawing
• ISSN: 0011-2240; 1090-2392
• DOI: 10.1016/j.cryobiol.2014.11.002

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 (80μM, 120μM, 160μM, 200μM, 240μM, or 320μM) for 45min to evaluate effects of dye dose. The frozen-thawed sperm were stained with 160μM Hoechst33342 for various durations (0min, 15min, 30min, 45min, 60min, 75min, or 90min) 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