Application of MTT reduction assay to evaluate equine sperm viability.
The assay of MTT reduction depends on the ability of metabolically active cells to reduce the tetrazolium salt (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) to formazan. This study was conducted to examine and validate of a simple and less costly MTT test in determining equine sperm viability and compare the efficiency of this test with a flow cytometer. Fresh ejaculates from 11 stallions (warm blood) were included in this study. Semen was diluted to 100 million cells/ml in a Hepes 0.1% BSA. The rates of MTT reduction were measured in microtiter plates after incubation for 1 and 4h at 37 degrees C using spectrophotometer (MS2 Reader) at wavelength 550 nm. Simultaneously split samples of the same semen were tested, using a flow cytometer for mitochondrial activity, sperm viability, and acrosomal integrity using Rhodamine 123, SYBR-14 and LysoTracker Green DNA-26, respectively. The results revealed a strong correlation (P < 0.001) between the results of MTT test at 1 and 4 h of incubation time and the result of mitochondrial activity (r = 0.978, 0.977), sperm viability (r = 0.954, 0.977) and acrosomal integrity (r = 0.867, 0.886). In conclusion, the MTT test was found to be a reliable method in evaluating semen viability and can be used successfully, especially in routine analysis, where practical aspects such as time, costs and practicability are important.
Aziz
D M
DM
Department of Surgery and Obstetrics, College of Veterinary Medicine, Mosul, Iraq. dhaferaziz@web.de
Ahlswede
L
L
Enbergs
H
H
D000177:Acrosome / Q000502:physiology*; D000818:Animals; D002470:Cell Survival; D004396:Coloring Agents; D005069:Evaluation Studies as Topic; D005434:Flow Cytometry / Q000379:methods / Q000662:veterinary; D006736:Horses; D008297:Male; D010084:Oxidation-Reduction; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000662:veterinary; D013081:Sperm Motility / Q000502:physiology*; D013094:Spermatozoa / Q000502:physiology*; D013997:Time Factors
D004396:Coloring Agents