The effects of ferulic acid, tryptophan, and L-Glutamine on mouse sperm freezability

In the present study, the activities of Ferulic acid (0.1mM, 1mM ve 10mM), Tryptophan (5mM, 25mM ve 50mM) and L-Glutamine (10mM, 50mM ve 100mM) at different doses added to 18% raffinose + 3% skimmed milk powder semen extender on the freezing of mouse spermatozoa cells were investigated. The group of 18% raffinose + 3% skimmed milk powder combination was used as the control group. In the study, 60 male CD-1 mice aged 12-24 weeks were used. Mice were divided into 10 groups, including one control group and nine experimental groups. Each group consisted of 6 mice. Mice were sacrificed by cervical dislocation method and spermatozoa were obtained from cauda epididymis. The spermatozoa obtained from the control group mice were diluted with a semen extender consisting of 18% raffinose + 3% skimmed milk powder. To the experimental groups, 18% raffinose + 3% skimmed milk powder semen extender was added to 0.1, 1 mM and 10 mM ferulic acid, 5 mM, 25 mM and 50 mM Tryptophan and 10 mM, 50 mM and 100 mM L-Glutamine. After incubating for 10 minutes, diluted semen was drawn into 0.25 ml straws and frozen in liquid nitrogen vapor. Frozen semen was thawed in a 37oC water bath for 30 seconds. After freezing thawing, motility, dead spermatozoa ratio, plasma membrane integrity, abnormal acrosome ratio, motility endurance and cell apoptosis tests were performed in HTF (Human Tubal Fluid) for 4 hours. Compared with the control group after freeze-thaw, the highest motility and plasma membrane integrity were obtained in the 10 mM L-Glutamine group with 56.67±2.11% and 77.83±0.87%, respectively (P<0.05). In addition, when compared to the control group, the lowest rate of dead spermatozoa and abnormal acrosome were found in the 10 mM L-Glutamine group as 26.00±1.46% and 6.33±1.09%, respectively (P<0.05). The duration of longevity after freezing and thawing was determined in the 10 mM and 50 mM L-Glutamine group up to the 4th hour compared to the control group (P<0.05). In the evaluation of apoptosis in semen samples, there was no significant difference between control, 0.1 mM Ferulic acid and 10 mM L-Glutamine groups (P>0.05). As a result, it was determined that the addition of 10 mM L-Glutamine to the semen extender increased the motility after freeze-thaw, viable spermatozoa, functional membrane integrity ratio, abnormal acrosome ratio or motility resistance and could be used successfully in mouse semen freezing extenders. Keywords: Mouse, frozen-thawed semen, ferulic acid, tryptophan, L-glutamine.