Prediction of Nili-Ravi buffalo bull fertility through Fourier harmonic analysis of sperm

Fourier harmonic analysis (FHA) is a robust method for identification of minute changes in sperm nuclear shape that are indicative of reduced fertility. The current study was designed to develop a fertility prediction model for Nili-Ravi buffalo bulls through FHA of sperm. In experiment I, FHA technique was standardized, average sperm nuclear perimeter was measured and sperm nuclear shape plot of buffalo bull was constructed. Sperm of buffalo bulls (n = 10) were stained with YOYO-1 and Hoechst -33342 to differentiate live and dead, and digital images were captured using phase contrast and fluorescent microscopy. The images were analyzed by ImageJ software and 100 sperm/bull were evaluated. The results are described as mean +/- SEM values of mean harmonic amplitude (mharm), skewness harmonic amplitude (skharm), kurtosis harmonic amplitude (kurharm) and variance harmonic amplitude (varharm) at Fourier frequencies 0 -5 along with the cartesian and polar coordinate plots of buffalo bull sperm. In experiment II, a fertility prediction model was developed based on FHA of buffalo bull sperm. Semen samples of low (n = 6), medium (n = 3) and high (n = 8) fertility bulls were investigated for FHA of sperm and harmonic amplitudes (HA) were generated. Firstly, to determine if live and dead sperm population have unique nuclear shape distribution; the mean, skewness, kurtosis and variance HA 0 -5 of 1700 live and 1294 dead spermatozoa of 17 bulls were evaluated. T -test signified a difference in the mharm0 (2.363 +/- 0.01 vs. 2.439 +/- 0.02), skharm0 (-0.0002 +/- 0.07 vs. -0.266 +/- 0.09), kurharm0 (-0.156 +/- 0.07 vs. 0.260 +/- 0.18), kurharm2 (0.142 +/- 0.11 vs. 1.031 +/- 0.32) and varharm4 (0.109 +/- 0.00 vs. 0.082 +/- 0.00) of live vs. dead sperm population (p < 0.05). Therefore, 100 live sperm/bull were further evaluated for mean, skewness, kurtosis and variance HA 0 -5 values among high (n = 6) and low -fertility (n = 6) groups. Results of T -test showed higher values of mharm2 (0.739 +/- 0.01 vs. 0.686 +/- 0.00), mharm4 (0.105 +/- 0.001 vs. 0.007 +/- 0.001), and skharm0 (0.214 +/- 0.109 vs. -0.244 +/- 0.097) in high vs. low -fertility group (p < 0.05). In next step, five significantly different combinations of discriminant measures between high and low -fertility groups were obtained by discriminant analysis. In conclusion, mharm4, skharm0 and varharm2 correctly identified 91.7 % of bulls into their respective fertility groups, and upon cross validation the value of the canonical correlation was 0.928.