Application of MTT reduction assay to evaluate equine sperm viability.
Abstract: 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.
Publication Date: 2005-04-02 PubMed ID: 16139611DOI: 10.1016/j.theriogenology.2005.02.009Google Scholar: Lookup
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- Comparative Study
- Journal Article
Summary
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This research paper explores the use of the MTT reduction assay, a simple and inexpensive test, to determine the viability of horse sperm. The study correlates the results of this test with those obtained through a flow cytometer, revealing a strong correlation.
Research Methodology and Experimentation
- This research used fresh samples of horse semen taken from 11 different warm blood stallions.
- The semen was diluted to a concentration of 100 million cells/ml in a Hepes 0.1% BSA.
- The MTT reduction rates were measured using spectrophotometer reading at wavelength 550 nm, after incubation of the samples for periods of 1 and 4 hours at a temperature of 37 degrees Celsius.
- Further tests on split samples of the same semen were carried out using a flow cytometer, allowing for comparison of results. These tests checked for mitochondrial activity, sperm viability, and acrosomal integrity using Rhodamine 123, SYBR-14, and LysoTracker Green DNA-26 respectively.
Results and Conclusion
- The research found a strong correlation between the results of the MTT test and those from the flow cytometer. Specifically, significant correlations were observed for sperm viability (r = 0.954, 0.977) and acrosomal integrity (r = 0.867, 0.886).
- The research found an even stronger correlation between mitochondrial activity (r = 0.978, 0.977) and the results of the MTT test.
- As a result, the study concluded that the MTT test is a reliable method for evaluating the viability of horse sperm.
- Given its simplicity and cost-effectiveness, the test is particularly suitable for routine analyses where factors like cost, time, and practicality are considerations.
Cite This Article
APA
Aziz DM, Ahlswede L, Enbergs H.
(2005).
Application of MTT reduction assay to evaluate equine sperm viability.
Theriogenology, 64(6), 1350-1356.
https://doi.org/10.1016/j.theriogenology.2005.02.009 Publication
Researcher Affiliations
- Department of Surgery and Obstetrics, College of Veterinary Medicine, Mosul, Iraq. dhaferaziz@web.de
MeSH Terms
- Acrosome / physiology
- Animals
- Cell Survival
- Coloring Agents
- Evaluation Studies as Topic
- Flow Cytometry / methods
- Flow Cytometry / veterinary
- Horses
- Male
- Oxidation-Reduction
- Sensitivity and Specificity
- Spectrophotometry / veterinary
- Sperm Motility / physiology
- Spermatozoa / physiology
- Time Factors
Citations
This article has been cited 7 times.- Li S, Ren J, Zhang W, Wang B, Ma Y, Su L, Dai Y, Liu G. Glutathione and selenium nanoparticles have a synergistic protective effect during cryopreservation of bull semen. Front Vet Sci 2023;10:1093274.
- Buranaamnuay K. The MTT assay application to measure the viability of spermatozoa: A variety of the assay protocols. Open Vet J 2021 Apr-Jun;11(2):251-269.
- Momeni HR, Abnosi MH, Eskandari N. Quantitative evaluation of human sperm viability using MTT assay: A laboratory study. Int J Reprod Biomed 2020 Nov;18(11):983-988.
- Chen YH, Wu CP, Lin HL, Liaw RB, Lai YY, Wu MC, Chen LR, Jason Tsai PS. Tetrazolium Salt WST-8 as a Novel and Reliable Chromogenic Indicator for the Assessment of Boar Semen Quality. Animals (Basel) 2020 Dec 4;10(12).
- Matsuura K, Wang WH, Ching A, Chen Y, Cheng CM. Paper-Based Resazurin Assay of Inhibitor-Treated Porcine Sperm. Micromachines (Basel) 2019 Jul 25;10(8).
- Eskandari F, Momeni HR. Protective effect of silymarin on viability, motility and mitochondrial membrane potential of ram sperm treated with sodium arsenite. Int J Reprod Biomed 2016 Jun;14(6):397-402.
- van den Berg BM. Microscopic analysis of MTT stained boar sperm cells. Open Vet J 2015;5(1):58-63.
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