To explore the effect of silymarin on bull spermatozoa during cooling and cryopreservation. Semen was collected by artificial vagina from Holstein bulls born in Iraq  and diluted by Tris-Citrate-Fructose egg yolk diluents. Diluted spermatozoa were treated with silymarin (0.018 mg/50ml, 0.027 mg/50ml, 0.036 mg/50ml), and packaged in 0.25 ml straws, which were further cooled to 5°C before freezing in liquid nitrogen. Thawing was carried out at 37°C for 30 sec, and the progressive motility%, dead%, abnormality% and acrosome integrity% were assessed, concentrations of addition to the control (0.0 mg/ml) reaching a final volume of 10 ml in each tube. The results proved after cooling and freezing, Individual motility% of sperm was significantly (p< 0.05) higher in T1 which differed significantly (p< 0.05) from T2 and T3 and all these values significantly (p< 0.05) different from control mean while values of dead sperm% show the lowest in T1 which is significantly (p< 0.05) different from control after cooling while after freezing lowest value in T1 and T2 and highest in T3 and control with significant (p<0.05) different. Abnormal sperm% was lowest in T1 after cooling with significant different(p< 0.05) from T2, T3 and control.

This study aims to identify whether adding silymarin and melatonin separately or together to the tris diluent in maintaining the fertile viability of the sperm after cooling and freezing in liquid nitrogen. Semen was collected by artificial vagina from Holstein bulls born in Iraq and diluted by Tris-Citrate-Fructose egg yolk diluents. Diluted spermatozoa was treated with tris diluent without any addition (control), silymarin 0.018gm/50ml tris (T1), combination of silymarin and melatonin (T2) and melatonin 0.05gm/100ml tris alone (T3), and packaged in 0.25 ml straws, which were further cooled to 5°C before freezing in liquid nitrogen, and the progressive motility%, dead sperm%, abnormality% and acrosome defects% were assessed after cooling and freezing. Thawing was carried out at 37°C for 30 sec. The results indicate that the addition of silymarin and melatonin had a role in improving and maintaining the sperm properties whether after cooling or freezing, but silymarin was better than melatonin alone or combination of them in reducing sperm damage after cooling and freezing. The results also showed that freezing had a negative effect on the sperm properties, but it was less or no effect of the diluent contain  silymarin alone.