ASSESSMENT OF VIABILITY AND MITOCHONDRIAL-FUNCTION OF EQUINE SPERMATOZOA USING DOUBLE STAINING AND FLOW-CYTOMETRY

An objective double-staining method was developed to evaluate viabilit y and mitochondrial function of stallion spermatozoa using flow cytome try. Sperm viability was assessed by propidium iodide (PI) exclusion, and mitochondrial function was measured by the intensity of rhodamine 123 (R123) fluorescence. Flow cytometry estimates of sperm viability m easured by PI were equivalent (P > 0.05) to estimates made using Hoech st 33258 stain and fluorescent microscopy (% dead: 25 +/- 2.4 vs 21.5 +/- 3.5). The use of both PI and R123 was validated by addition of var ious proportions of freeze-shocked (membrane damaged) cells to viable spermatozoa. There was a high correlation (r(2) = 0.996) between incre ased PI positive-stained (dead) cells and the number of membrane-damag ed spermatozoa added (46 dead: 29 +/- 0.4, 44 +/- 1.4, 58 +/- 0.9, 75 +/- 0.7 and 91 +/- 0.25 vs 0, 25, 50, 75 and 100% damaged cells, respe ctively). Optimal mitochondrial activity (OMA), as assessed by R123 up take, was also reduced proportionally (r(2) = 0.976) by the percentage of membrane-damaged cells added (% OMA: 48 +/- 0.6, 37 +/- 1.7, 29 +/ - 0.5, 16 +/- 1, 3.8 +/- 1.3 vs 0, 25, 50, 75 and 100% damaged cells, respectively). The mitochondrial inhibitors rotenone and monensin sign ificantly depressed optimal mitochondrial activity (P < 0.001), and th ere was a significant positive correlation (r(2) = 0.959) between the dose of inhibitors added and the population of sperm cells exhibiting minimal R123 staining (4 +/- 0.9, 12 +/- 1.6, 14 +/- 0.1 and 28 +/- 2% for treatments with 0, 0.5, 1 and 2 x 10(-5) M rotenone and 0, 0.5, 1 , and 2 x 10(-4) M monensin, respectively). Finally, it was shown that treatments containing identical proportions of membrane-damaged cells yielded similar results in terms of viability and mitochondrial activ ity, irrespective of whether the staining procedure was single or doub le (P > 0.05). The results of the double-staining method revealed that the percentage of spermatozoa with optimally functioning mitochondria was significantly correlated with the percentage of viable (PI negati ve) sperm cells (r(2) = 0.998). Flow cytometric analyses using this st aining procedure provides reliable and rapid (10,000 cells/min) qualit ative assessment of stallion semen. (C) 1997 by Elsevier Science Inc.