Simultaneous evaluation of plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential in bovine spermatozoa by flow cytometry

被引：5
|
作者：
Kanno, Chihiro [1 ]
Kang, Sung-Sik [1 ]
Kitade, Yasuyuki [1 ]
Yanagawa, Yojiro [1 ]
Takahashi, Yoshiyuki [1 ,3 ]
Nagano, Masashi [1 ,2 ]
机构：
[1] Hokkaido Univ, Grad Sch Vet Med, Dept Vet Clin Sci, Lab Theriogenol, Sapporo, Hokkaido 0600818, Japan
[2] Hokkaido Univ, Grad Sch Vet Med, Dept Vet Clin Sci, Lab Theriogenol,Kita Ku, Kita 18 Nishi 9, Sapporo, Hokkaido 0600818, Japan
[3] Genet Hokkaido Assoc, Sapporo, Hokkaido 0600040, Japan
来源：
ZYGOTE | 2016年 / 24卷 / 04期
基金：
日本学术振兴会;
关键词：
Acrosomal integrity; Bovine spermatozoa; Flow cytometry; Mitochondrial membrane potential; Spermatozoon viability; MAMMALIAN SPERM; SEMEN QUALITY; FERTILITY; CRYOPRESERVATION; VIABILITY; PROBES;
D O I：
10.1017/S0967199415000490
中图分类号：
Q2 [细胞生物学];
学科分类号：
071009 ; 090102 ;
摘要：
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.
引用
收藏
页码：529 / 536
页数：8
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