Evaluation of Boar Sperm Viability by MTT Reduction Assay in Beltsville Thawing Solution Extender *

MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) reduction assay is a method that validates the viability of an active cell. Dehydrogenase in mitochondria converts yellow colored insoluble tetrazolium salt to purple colored watersoluble formazan. Sperm also have mitochondria in the midpiece, therefore sperm viability could be evaluated by MTT reduction assay. Several studies have already demonstrated the capability of application of the MTT reduction assay to sperm of several species in Hepes-BSA buffer. Because most liquid semen was diluted in extender like BTS (Beltsville Thawing Solution), Modena or Androhep when it is used or transferred, semen needed another dilution in Hepes-BSA buffer to assess sperm viability. In this study, we evaluated boar sperm viability especially in BTS extended semen and compared the efficiency of this test with eosin-nigrosin staining. We used the fresh BTS extended semen from a local A.I center. Semen sample was diluted to 3.0×10 sperms/ml in BTS. The rates of formazan production were measured in 96-well microtiter plates immediately and 1h after incubation at 17°C using a spectrophotometer at wave length 560 nm. Simultaneously, split samples of the same semen were tested, using eosin-nigrosin staining to compare the efficiency of the MTT assay of sperm viability in BTS. The correlation between the results of these tests was calculated using Student-t test and ANOVA. The results revealed a strong correlation between the results of MTT reduction rate and the results that were simultaneously determined by eosin-nigrosin staining at 1 h. In conclusion, the MTT reduction test was an effective and simple method to validate sperm viability and it could be used as a simple tool to evaluate sperm viability in the local A.I center and laboratory. (


INTRODUCTION
There are several methods for evaluating sperm viability.Visual estimation of viable sperm using microscope is commonly used for it.Eosin-nigrosin method and HOST (Hypo-Osmotic Swelling Test) are simple and inexpensive; however the result can be influenced by experience of analyst (McNiven et al., 1992;Nasr-Esfahani et al., 2002;Jang et al., 2006).By contrast, CASA (Computer Assisted Sperm Analysis) and flow cytometry are the more objective method.CASA measure the speed of sperm and abnormality of shape.It uses the computer and camera to measure these characters of sperm (Arman et al., 2006).
Flow cytometry is a technology that allows a single cell to be measured for a variety of characteristics.It is different technical applications takes many advantages for the analysis of sperm quality.Because flow cytometry evaluates individual cell with rapid speed, it is objective and accurate method (Cordelli et al., 2005).
MTT (3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyltetrazolium bromide) is a yellow water-soluble tetrazolium salt.Succinate dehydrogenase system of active mitochondria converts this dye to water-insoluble purple formazan on the reductive cleavage of its tetrazolium ring (Slater et al., 1963).Thus, the amount of formazan formed can be determined spectrophotometrically and serves as an estimate of the number of mitochondria and hence the number of living cells in the sample (Denizot and Lang, 1986;Song et al., 2007).In many studies, MTT assay was used which were related to viability of different cells (Mosmann, 1983; Levitz and Diamone, 1985; Carmichael et  al., 1987; Campling et al., 1988; Freimoser et al., 1999).Therefore, this method can be used to assess sperm viability and MTT reduction assay already have successfully applied to check sperm viability in many species (Capkova et al., 2000;Park et al., 2002Park et al., , 2004;;Aziz et al., 2005;Hu et al., 2006).In case of boar semen, no research has been studied.Fresh boar semen sample is diluted in BTS (Beltsville Thawing Solution) extender.BTS extender is commonly used to dilution the fresh boar semen for transfer and storage (Joshi et al., 2006).
In this study, we evaluated the boar sperm viability especially in BTS extender using simple and less costly MTT reduction assay and compare the efficiency with eosin-nigrosin staining.

Semen samples and analysis
Liquid semen which was diluted in BTS (at 30×10 6 sperm/ml) were used in this study from Local A.I center.To evaluate sperm viability, 1% eosin-nigrosin stain method was performed according to Björndahl's method (2003).The staining solution for the one-step eosin-nigrosin staining contained 0.67 g eosin Y, 0.9 g sodium chloride and 10 g of nigrosin.Aliquots of each 6 samples (50 μl) mixed with same amount of solution in micro tube.The suspension was incubated for 30 s at room temperature.Then, a 12 μl droplet was transferred into slide where it was smeared by sliding a cover slip.After smear the samples, dried few second and examine directly 250 sperm were assessed three times at a magnification 400× in each samples.

MTT reduction assay and experiments
The MTT assay was performed by routine methods (Mosmann, 1983;Aziz et al., 2005).For each sample, 6 wells of the 96-well microplate were used.One hundred microliters of semen sample plus 10 μl of MTT stock solution (5 mg of MTT/ml of PBS) were placed in each well.The optical density of samples were measured immediately and after 1 h of incubation at 17°C using a spectrophotometer (μ Quant TM Microplate Spectrophotometer, Bio-Tek instruments, USA) at a wave length of 560 nm.MTT reduction rate (optical density) for each sample was calculated by concuring the difference between the first and second reading of the spectrophotometer.
The freeze-killed procedure was used, in order to obtain the standard curve and the relationship between the MTT reduction rate and sperm viability.Liquid semen samples of Duroc with good quality (about 80% viable sperms) were used.After the dilution of Semen with BTS, 6 ml of the diluted semen divided in two fractions; one fraction was maintained at 17°C, while the sperms in the other fraction were killed by two cycles of plunging into liquid nitrogen and thawing at 37°C.Samples for analysis were made by combining aliquots of viable and freeze-killed sperms at ratios of 10:0, 8:2, 6:4, 4:6, 2:8 and 0:10 v/v, respectively.The prepared samples were analyzed by (1) MTT and spectrophotometer, and (2) Eosin-nigrosin stain.MTT reduction rates were determined immediately and each hour up to 4h incubation at 17°C.
The MTT test was applied to evaluate the sperm viability in liquid semen of Duroc.After the dilution of semen samples in BTS, samples and MTT stock solution were distributed in the wells of the 96-wll microplate.The rates of MTT reduction were taken immediately, 1 and 4 h after incubation at 17°C.Simultaneously split samples of the same semen were tested using eosin-nigrosin stain method to evaluate the efficiency of MTT reduction assay in assess the sperm viability.

Statistic analysis
Regression analysis was used to evaluate the efficiency of the MTT test for the assessment of sperm viability of boar semen.Data was analyzed using KCjunior (Bio-Tek autoreader control Version 1.41.8), and p<0.05 was considered as statistically significant.

Analysis of the results
Initial results showed MTT reduction rates of the semen sample groups which contained different proportions of viable sperm in Figure 1.The rate of MTT reduction assay increased gradually with incubation time, in all groups.Increasing the volume of viable sperm cells in semen samples resulted in a proportional and significant increase in the rate of MTT reduction.At each time of measurement, the MTT reduction rate correlated (p<0.001,r>0.915) positively with proportion of freeze-killed dead sperms.
The color changes of MTT from yellow to purple was very clear after 1 h of the incubation time and up to 4 h, especially in midpiece are shown Figure 2. Two standard curves (at 1 h, y = 1.7545x-0.1684and 4 h, y = 0.8268x-0.0804)for the relationship between the MTT reduction rate and the percentage of viable sperms were acquired (Figures 3 and 4).These two standard curves were applied later to acquire the percentage of viable sperm cells in each sample in accordance with the MTT reduction rate.
The results of MTT reduction rate and that of eosinnigrosin stain are shown in Table 1.The results showed a high correlation (p<0.001) between the results of MTT test at 1 and 4 h of incubation time and the results of eosinnigrosin stain which evaluated the sperm viability.The MTT reduction rate was significantly (p<0.001)correlated with the results that simultaneously determined by eosinnigrosin staining, yielding correlation coefficients of r = 0.9493 for 1 h and r = 0.9564 for 4 h.

Discussion and further study
The MTT assay was used in many studies to evaluate the viability of different cells.This test depends on the ability of viable cells to convert the MTT to purple formazan.In this study, the diagnostic value of the MTT reduction assay to evaluate the boar sperm viability was investigated by comparison of other method which is already believed reliable, that is, eosin-nigrosin staining especially in BTS extended semen (Zhou et al., 2004).
In contrast to the procedure that was published previously, the MTT reduction rate was taken successfully after 1 h of incubation time.This is expected because spermatozoa are very active cells and rich in mitochondria; therefore, the reduction of MTT by spermatozoa is faster   than other cells.A similar observation was reported in other previous study in equine and bovine (Aziz et al., 2005;Aziz, 2006).Because other studies have already revealed that sperm viability is positively related to sperm quality parameters like acrosome integrity, mitochondrial activity and even these parameters also correlate positively with fertility (Garner et al., 1997), these findings is useful in evaluating liquid boar semen quality.Furthermore, we observed similar results with other experiments in BTS extended semen, so liquid semen wouldn't need any other treat which can affect sperm viability.
The advantages of the MTT test are that it is feasible and reproducible (Ciapetti et al., 1992).Additionally, results from this study suggest other advantages of this test in evaluating the bovine semen.Firstly, this test is fast (1 h); secondly, many samples (up to 10) can be examined at the same time; these test wouldn't need any other treat.When we would apply this method to assess sperm viability in laboratory or local A.I center, some problems seems to have to be solved.Firstly, we have to plate same number of sperm in testing well; secondly, identify whether the formazan affect sperm viability or DNA.The MTT reduction test was effective and simple method to validate sperm viability and it would be used simple tool to evaluate sperm viability in local A.I center and laboratory.

Figure 1 .
Figure1.MTT reduction rates of the semen samples containing different portion of live and dead sperms.The optical density of samples were measured immediately and after 1h of incubation at 17°C using a spectrophotometer (μQuant TM Microplate Spectrophotometer, Bio-Tek instruments, USA) at a wave length of 560 nm.

Figure 3 .Figure 4 .
Figure3.Linear regression between the MTT reduction rate at 1 h of incubation time and the percentage of viable sperm, which was evaluated by eosin-nigrosin staining.Data was analyzed using KCjunior (Bio-Tek autoreader control Version 1.41.8), and p< 0.05 was considered as statistically significant.

Table 1 .
Analysis of the liquid semen samples using MTT test and eosin-nigrosin staining for evaluating viability