Assessment of Nili-Ravi buffalo (Bubalus bubalis) semen by MTT reduction assay

MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay is commonly used to validate the viability of metabolically active cells. The study was conducted to examine and validate the MTT test to assess the sperm viability of Nili-Ravi buffalo bulls and compare the efficiency of the test with the supra-vital staining technique (eosin-nigrosine) and hypo-osmotic swelling test. Fresh semen samples from breeding Nili-Ravi buffalo bulls (n = 20) were collected using an artificial vagina. After assessing the quality of semen for normal parameters, the MTT assay was carried out in phosphate buffer saline. Results revealed a high significant correlation (r = 0.995) between the viability of sperm and the rate of reduction of MTT. The other proportions of some semen samples showed a weak relationship between the eosinnigrosine method (r = -0.32), hypo-osmotic swelling test (r = -0.12) and motility (r = -0.08). However, the MTT assay was found to be superior to other tests as it was able to determine those sperm which were more than 90% viable. In conclusion, the MTT assay is a simple, robust test that can be used to select Nili-Ravi buffalo bulls on the basis of sperm quality.