Spectrophotometric calibration and comparison of different semen evaluation methods in nili-ravi buffalo bulls

Abstract

This study was designed with the aim to find suitable spectrophotometric wavelength for accurate measurement of sperm concentration in Nili Ravi buffalo and to compare different quantitative methods (visual, hemocytometric and spectrophotometric) for the assessment of sperm concentration. A total of 78 fresh semen ejaculate were collected from breeding buffalo bulls, diluted with required concentrations and relationship between sperm number and absorbance was determined at 350, 450, 546 and 650 nm wavelengths by using spectrophotometer. The spectrometric results showed that λmax was obtained at 350 nm. The derived equation between sperm concentration counted by haemocytometer and absorbance at 350 nm was Y = 0.1135x + 0.0002 (R2 = 0.9924), at 450 nm, Y = 0.1011x-0.0436 (R2 = 0.9756), at 546nm, Y = 0.0825x-0.0101 (R2 = 0.9603) and at 650 nm, Y = 0.0774x-0.0235 (R2 = 0.9602). These results were later compared with relative sperm numbers at different dilutions. The results showed that 350nm wavelength appeared suitable for estimation of sperm concentration compared to others. Additionally, it was found that both photometer(P<0.01) and haemocytometer (P<0.01) significantly counted more sperm cells than visual assessment as used regularly on different SPUs. Moreover, haemocytometer counted significantly (834×106Vs 678×106, P=0.005) more sperm cell than Photometer. Finally, regression analysis between haemocytometer and Photometer showed significant slope of regression and regression coefficient was 0.771 which warns that photometer must be calibrated before proper employment for routine measurement of sperm concentration.