title: Simultaneous evaluation of plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential in bovine spermatozoa by flow cytometry.
abstract: The present study aimed to develop an objective evaluation procedure to estimate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bull spermatozoa simultaneously by flow cytometry. Firstly, we used frozen-thawed semen mixed with 0, 25, 50, 75 or 100% dead spermatozoa. Semen was stained using three staining solutions: SYBR-14, propidium iodide (PI), and phycoerythrin-conjugated peanut agglutinin (PE-PNA), for the evaluation of plasma membrane integrity and acrosomal integrity. Then, characteristics evaluated by flow cytometry and by fluorescence microscopy were compared. Characteristics of spermatozoa (viability and acrosomal integrity) evaluated by flow cytometry and by fluorescence microscopy were found to be similar. Secondly, we attempted to evaluate the plasma membrane integrity, acrosomal integrity, and also mitochondrial membrane potential of spermatozoa by flow cytometry using conventional staining with three dyes (SYBR-14, PI, and PE-PNA) combined with MitoTracker Deep Red (MTDR) staining (quadruple staining). The spermatozoon characteristics evaluated by flow cytometry using quadruple staining were then compared with those of staining using SYBR-14, PI, and PE-PNA and staining using SYBR-14 and MTDR. There were no significant differences in all characteristics (viability, acrosomal integrity, and mitochondrial membrane potential) evaluated by quadruple staining and the other procedures. In conclusion, quadruple staining using SYBR-14, PI, PE-PNA, and MTDR for flow cytometry can be used to evaluate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bovine spermatozoa simultaneously.
authors.affiliation: Laboratory of Theriogenology,Department of Veterinary Clinical Sciences,Graduate School of Veterinary Medicine,Hokkaido University,Sapporo 060-0818,Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Kanno
authors.affiliation: Laboratory of Theriogenology,Department of Veterinary Clinical Sciences,Graduate School of Veterinary Medicine,Hokkaido University,Sapporo 060-0818,Japan.
authors.forename: Sung-Sik
authors.initials: SS
authors.lastname: Kang
authors.affiliation: Laboratory of Theriogenology,Department of Veterinary Clinical Sciences,Graduate School of Veterinary Medicine,Hokkaido University,Sapporo 060-0818,Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Kitade
authors.affiliation: Laboratory of Theriogenology,Department of Veterinary Clinical Sciences,Graduate School of Veterinary Medicine,Hokkaido University,Sapporo 060-0818,Japan.
authors.forename: Yojiro
authors.initials: Y
authors.lastname: Yanagawa
authors.affiliation: Laboratory of Theriogenology,Department of Veterinary Clinical Sciences,Graduate School of Veterinary Medicine,Hokkaido University,Sapporo 060-0818,Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Takahashi
authors.affiliation: Laboratory of Theriogenology,Department of Veterinary Clinical Sciences,Graduate School of Veterinary Medicine,Hokkaido University,Kita-ku Kita 18 Nishi 9,Sapporo 060-0818,Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Nagano
mesh_terms: D000177:Acrosome / Q000502:physiology*; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology*; D015925:Cryopreservation / Q000379:methods; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000502:physiology*; D008856:Microscopy, Fluorescence; D009930:Organic Chemicals / Q000737:chemistry; D011419:Propidium / Q000737:chemistry; D015203:Reproducibility of Results; D012662:Semen Preservation; D013094:Spermatozoa / Q000502:physiology*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D009930:Organic Chemicals; C093207:SYBR-14; D011419:Propidium
keywords: Acrosomal integrity; Bovine spermatozoa; Flow cytometry; Mitochondrial membrane potential; Spermatozoon viability