Assessment of bovine sperm viability by MTT reduction assay

The MTT reduction assay 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 a simple and less costly MTT test to determine bovine sperm viability and compare the efficiency of this test with a flow cytometer. Fresh ejaculates from eight bulls were included in this study. Semen sample was diluted to 30x10(6) sperms/ml in a Hepes 0.1% BSA. The rates of MTT reduction were measured in microtiter plates after incubation for 1h at 37 degrees C using spectrophotometer (MS2 Reader) at wave length 550nm. Simultaneously split samples of the same semen were tested, using a flow cytometer for sperm viability, mitochondrial activity, and acrosomal integrity using SYBR-14, Rhodamine 123 and LysoTracker Green DNA-26, respectively. The correlation between the results of these tests was calculated using the Pearson correlation coefficients. The results revealed a strong correlation (P<0.001) between the results of MTT reduction rate and the results that simultaneously determined by flow cytometer, yielding correlation coefficients of r=0.950 for sperm viability, of r=0.926 for mitochondrial activity and of r=0.959 for acrosomal integrity. The same correlation coefficient was observed between the values of sperm viability calculated on the basis of MTT reduction rates and the results of flow cytometer. In conclusion, the MTT reduction test was found to be a reliable method in evaluating bovine semen viability and can be used successfully, especially in routine analysis, where practical aspects such as time, costs and practicability are important.     

A novel automated fluorometric assay to evaluate sperm viability and fertility in dairy bulls

The artificial insemination (AI) industry is in need of an objective and rapid, but inexpensive method to evaluate frozen thawed bull semen ejaculates. This study presents a new fluorescence method that uses an automatized fluorometer and fluorophore stain propidium iodide that stains only those cells with damaged membranes. The fluorescence of the semen sample and the totally killed subsample were measured simultaneously, and viability was calculated. Every semen batch was analyzed before use in AI. For fertility evaluation, the nonreturn rates (NR%) obtained from 92,120 inseminations with the analyzed batches were recorded from 166 bulls (436 batches). This study confirms a 3.9% better NR% for the Finnish Holstein-Friesian breed than for Finnish Ayrshire. There was a clear seasonality in NR%: it differed (5.3%) significantly, being best in summer to autumn (June to October) and lowest in winter (January to March). The fluorometer method was fast and easy. The correlation between the total number of viable spermatozoa in an insemination dose and field fertility was low but significant (r = 0.051, P = 0.016), suggesting that the plasma membrane integrity evaluation can serve as a cost-beneficial quality control method of frozen-thawed semen at bull stations.     

绿僵菌孢子活性的MTT比色法快速检测技术研究

通过比较孢子浓度、MTT终浓度、反应温度、反应时间、MTTf提取时间以及pH值等因素对孢子活性检测的影响,优化了MTT比色法杀蝗绿僵菌孢子活性检测条件,建立起稳定、灵敏、可靠的生化检测体系,适用于绿僵菌等真菌农药原药与制剂中活孢率的快速测定,为真菌农药研制提供了新的质量分析方法。     

Disruption of functional activity of mitochondria during MTT assay of viability of cultured neurons

The MTT assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in the cell cytoplasm to a strongly light absorbing formazan is among the most commonly used methods for determination of cell viability and activity of NAD-dependent oxidoreductases. In the present study, the effects of MTT (0.1 mg/ml) on mitochondrial potential (ΔΨ_m), intracellular NADH, and respiration of cultured rat cerebellum neurons and isolated rat liver mitochondria were investigated. MTT caused rapid quenching of NADH autofluorescence, fluorescence of MitoTracker Green (MTG) and ΔΨ_m-sensitive probes Rh123 (rhodamine 123) and TMRM (tetramethylrhodamine methyl ester). The Rh123 signal, unlike that of NADH, MTG, and TMRM, increased in the nucleoplasm after 5-10 min, and this was accompanied by the formation of opaque aggregates of formazan in the cytoplasm and neurites. Increase in the Rh123 signal indicated diffusion of the probe from mitochondria to cytosol and nucleus due to ΔΨ_m decrease. Inhibition of complex I of the respiratory chain decreased the rate of formazan formation, while inhibition of complex IV increased it. Inhibition of complex III and ATP-synthase affected only insignificantly the rate of formazan formation. Inhibition of glycolysis by 2-deoxy-D-glucose blocked the MTT reduction, whereas pyruvate increased the rate of formazan formation in a concentration-dependent manner. MTT reduced the rate of oxygen consumption by cultured neurons to the value observed when respiratory chain complexes I and III were simultaneously blocked, and it suppressed respiration of isolated mitochondria if substrates oxidized by NAD-dependent dehydrogenases were used. These results demonstrate that formazan formation in cultured rat cerebellum neurons occurs primarily in mitochondria. The initial rate of formazan formation may serve as an indicator of complex I activity and pyruvate transport rate.     

Relationship of MTT reduction to stimulants of muscle metabolism

MTT, a positively charged tetrazolium salt, is widely used as an indicator of cell viability and metabolism and has potential for histochemical identification of tissue regions of hypermetabolism. In the present study, MTT was infused in the constant-flow perfused rat hindlimb to assess the effect of various agents and particularly vasoconstrictors that increase muscle metabolism. Reduction of MTT to the insoluble formazan in muscles assessed at the end of experiments was linear over a 30 min period and production rates were greater in red fibre types than white fibre types. The vasoconstrictors, norepinephrine (100 nM) and angiotensin (10 nM) decreased MTT formazan production in all muscles but increased hindlimb oxygen uptake and lactate efflux. Veratridine, a Na(+) channel opener that increases hindlimb oxygen uptake and lactate efflux without increases in perfusion pressure, also decreased MTT formazan production. Membrane stabilizing doses (100 microM) of (+/-)-propranolol reversed the inhibitory effects of angiotensin and veratridine on MTT formazan production. Muscle contractions elicited by stimulation of the sciatic nerve, reversed the norepinephrine-mediated inhibitory effects on MTT formazan production, even though oxygen consumption and lactate efflux were further stimulated. Stimulation of hindlimb muscle oxygen uptake by pentachlorophenol, a mitochondrial uncoupler, was not associated with alterations in MTT formazan production. It is concluded that apart from muscle contractions MTT formazan production does not increase with increased muscle metabolism. Since the vasoconstrictors angiotensin and norepinephrine as well as veratridine activate Na(+) channels and the Na(+)/K(+) pump, energy required for Na(+) pumping may be required for MTT reduction. It is unlikely that vasoconstrictors that stimulate oxygen uptake do so by uncoupling respiration.     

Cell-based quantitative evaluation of the MTT assay

OBJECTIVE: To analyze the bioreduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) on a per cell basis and evaluate its modulation as a function of different stages of cell metabolism. STUDY DESIGN: Following MTT bioreduction, total optical density (TOD), cell area and specific activity (TOD/area) of V79 cells and cultured macrophages were recorded for individual cells by means of digital image analysis. The effect of different serum (0-10% vol/vol) or genistein (0-100 microM) concentrations was used to modulate the MTT-specific activity response. RESULTS: As cells in culture are heterogeneous in cell size, the contribution of each cell to the total amount of formazan formed per dish is variable. The production of formazan per cell as a result of MTT bioreduction was found to be proportional to cell size. CONCLUSION: Specific MTT-reducing activity was analyzed in phagocytes and nonphagocyte cells, revealing the utility of this variable in evaluating the MTT assay at the single-cell level.     

Females suffer a reduction in the viability of stored sperm following an immune challenge

Despite the ubiquitous nature of sperm storage in invertebrates, relatively little is known about its costs, or the impact that immune activation can have on a female's ability to maintain viable sperm stores. We explored the effects of an immune challenge on sperm storage under food‐limited and ad libitum conditions in the field cricket, Teleogryllus oceanicus, by injecting mated adult females with either a LD₅ dose of live bacteria or a nonpathogenic immune elicitor [lipopolysaccharide (LPS)] and then scoring the viability of their stored sperm. Females that were infected with bacteria showed a reduction in the viability of stored sperm 48 h after infection; interestingly, this pattern was not evident when females were injected with LPS. Reduction in sperm viability post‐infection may reflect a reproductive trade‐off between immune function and sperm store maintenance, as only females injected with bacteria showed an elevated antibacterial immune (lytic) response. Alternatively, bacteria may act directly on sperm quality. Dietary manipulations showed that lytic activity in females is condition dependent, irrespective of their immune challenge treatment. Diet affected the ability of females to maintain the viability of stored sperm, suggesting that sperm storage is condition dependent. That bacterial infection associated with a reduction in stored sperm quality has potentially important implications for the outcomes of sperm competition in T. oceanicus and in other species in which females store sperm between matings.     

Determinants of MTT reduction in rat hepatocytes

The determinants of reduction of the dye MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) in rat hepatocytes have been investigated. NADH, NADPH, and succinate were substrates for MTT reduction in rat liver homogenate, activity being greatest with NADH and least with succinate. Similar results were obtained with submitochondrial particles isolated from rat liver. NAD(P)Hdependent reduction of MTT was also detected in rat liver microsomes and cytosol. Rotenone, at a concentration that inhibited NAD(P)H-dependent MTT reduction in submitochondrial particles, did not inhibit MTT reduction in rat hepatocytes. Malonate, at a concentration that inhibited succinate-dependent MTT reduction in liver homogenate, did not inhibit MTT reduction in rat hepatocytes. Incubation of rat hepatocytes with ethanol or lactate (increase NADH levels), dicoumarol (inhibitor of DT-diaphorase), aminopyrine or hexobarbitone (substrates for the NADPH-requiring cytochrome P450-dependent microsomal monooxygenase) led to significant increases in the level of cellular MTT reduction. From these data, it is concluded that extramitochondrial NAD(P)H is the principal reductant for MTT reduction in rat hepatocytes, with mitochondrial dehydrogenase activity being only a minor contributor. It is also possible that cellular generation of superoxide (as might be expected on redox cycling of endogenous quinones following inhibition of DT diaphorase by dicoumarol) may be another source of MTT reduction. Caution should be exercised in ascribing an alteration in the level of cellular MTT reduction to a change in mitochondrial performance in the absence of corroborating evidence.     

用改良的MTT法测定rhG-CSF活性

ＭＴＴ测定法是根据线粒体脱氢酶催化ＭＴＴ形成蓝色甲■的多少来检测活细胞数和功能状态的,但原始方法中存在着一些问题,如敏感性偏低、有机溶剂产生蛋白质沉淀以及产物的溶解度偏低等。为了摸索测定 ｒｈＧ－ＣＳＦ活性的最适条件,我们以 ＮＦＳ－６０细胞为对象,比较了多种溶解缓冲液,并且对细胞数、ＭＴＴ浓度及保留时间、溶解液用量等条件进行了选择。结果表明,ＤＭＦ－２０％ＳＤＳ和 ２０％ＳＤＳ的效果最好,测定时细胞数为每孔 １０００个细胞,所加 ＭＴＴ浓度为 １ｍｇ／ｍｌ,保留时间为 ４ ｈ,溶解液的用量为每孔 １００μｌ。     

测定昆虫细胞存活或死亡的MTT方法的改进

MTT方法具有灵敏、简便、稳定可靠、不需同位素等特点。为了摸索测定昆虫细胞存活（或死亡）的MTT方法的各种最适条件,本文以中国棉铃虫细胞系为对象,确定了一些基本参数、并比较了三种MTT测定方法。结果表明：改进的方法所选用的溶剂——pH为4.5的3％SDS异丙醇效果最好,溶解甲所需时间短、OD值高。测定昆虫细胞范围为每孔500～60000细胞,测试波长为560nm,参考波长为690nm.     

An improved 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay for evaluating the viability of Escherichia coli cells

To reduce the interference in MTT reduction assay, we developed an improved protocol for evaluating the viability of Escherichia coli cells by conducting MTT reduction in 1.5-mL centrifuge tubes and formazan dissolution in test tubes. Our study is helpful in developing protocols for measuring the viability of other gram-negative bacteria.     

Coulter counter-based evaluation of sperm volume to assess sperm viability of bull semen and application to X/Y sperm sorting

The Coulter Counter Hypo-Osmotic Swelling test (CC-HOS) was developed to provide insight into the membrane integrity (relative volume shift Vr) of sperm necessary for fertilization, and to identify the optimum buffer needed for the X/Y chromosome sorting process. Using the CC-HOS test on neat bovine semen, the mean relative volume shift Vr for July and August was 1.20 and 1.14, respectively, whereas mean Vr values ranged from 1.32 to 1.41 during September to November. There was an inverse relationship between Vr magnitude and environmental temperature; we inferred that this enhanced sperm viability during autumn relative to summer. A method was developed to measure the dynamics of volume change of sperm in the buffer (pH 6.5) used for the X/Y chromosome sorting process. When exposed to the buffer (4 mM K+, 153 mM Na+, 140 mM Cl(-)), sperm from Bull C had a mean modal volume of 22.8+/-0.2 fL during a 0-300 s time interval, which did not significantly vary from sperm volumes (21.88+/-0.66 fL for Bull A and 22.46+/-0.38 fL for Bull B) noted in isotonic Isoton II solution. However, when exposed to lower ionic concentrations (2 mM K+, 62 mM Na+, 47 mM Cl-), the mean volume of Bull C sperm increased to 29.2+/-1.5 fL and exhibited slower rates toward stabilized volumes relative to higher ionic concentration buffers. Utilization of volume swelling measurements for measuring the impact of ion concentrations in X/Y chromosome sorting process buffers illustrated the importance of its application for emerging sperm-based biotechnologies.     

Effects of sperm conjugation and dissociation on sperm viability in vitro

Sperm conjugation is an unusual variation in sperm behavior where two or more spermatozoa physically unite for motility or transport through the female reproductive tract. Conjugation has frequently been interpreted as sperm cooperation, including reproductive altruism, with some sperm advancing their siblings toward the site of fertilization while ostensibly forfeiting their own ability to fertilize through damage incurred during conjugate break-up. Conversely, conjugation has been proposed to protect sensitive regions of spermatozoa from spermicidal conditions within the female reproductive tract. We investigated the possibility of dissociation-induced sperm mortality and tested for a protective function of conjugation using the paired sperm of the diving beetle, Graphoderus liberus. Sperm conjugates were mechanically dissociated and exposed to potentially damaging tissue extracts of the female reproductive tract and somatic tissue. We found no significant difference in viability between paired sperm and dissociated, single sperm. The results further indicate that the reproductive tract of female G. liberus might not be spermicidal and conjugation is not protective of sperm viability when damaging conditions do exist. Our results support the interpretation that, at least in some taxa, sperm conjugation is neither protective nor damaging to sperm viability.     

The hypoosmotic swelling test: Its employment as an assay to evaluate the functional integrity of the frozen-thawed bovine sperm membrane

The objective of this study was to determine the effectiveness of the hypoosmotic swelling (HOS) test together with the supravital test as a means of evaluating the functional integrity of frozen-thawed bovine sperm membrane. A solution consisting of equal parts of fructose and sodium citrate was prepared and the osmolality varied from 50 to 300 mOsm/L. From these various solutions under study, the 100 mOsm/L solution resulted in a maximal number of clearly identifiable swollen spermatozoa. The results from the supravital test indicated that the HOS solution preserved the integrity and prevented excessive lysis of the sperm membrane during the assay. A good correlation was found between the percentage of motile spermatozoa and spermatozoa that reacted to the HOS test (r = 0.73) and between the percentage of sperm with intact membranes and HOS reactive sperm (r = 0.81). Spermatozoa showing swelling of the entire tail region accounted for more than 60% of the total swelling for the HOS solution at 100 mOsm/L. The results obtained in this study indicate that frozen-thawed bovine spermatozoa did react to the HOS test. This technique could prove useful in studies involving the function of the sperm membrane and could possibly predict the sperm's ability to fertilize.     

MTT方法评价微生物细胞活性的探讨

对MTT比色法用于评价微生物细胞活性进行了探讨。本文以大肠杆菌为模式菌株,研究了不同浓度MTT、不同用量、在不同时间对试验结果OD570值的影响,结果表明细菌数在4.9×107－4.9×108个/mL范围内测出的OD570值与细菌浓度呈良好的正相关,0.5 mg/mL MTT用量20 &#61549;L,反应时间20 min时效果最佳,其相关回归方程为y = 0.1769x + 0.03,R2 = 0.9983。