The most important factor that leads to infertility
during the preservation of sperm is the peroxidation of
spermatozoon membrane lipids. Free radicals such as
superoxide radical, hydrogen peroxide and hydroxyl
radical react with cell membranes and cause oxidative
damage
16. The disruption of membrane integrity also
leads to the permeability of the cell to electrolytes. The
inclusion of calcium and partial sodium ions into the cell
destroys the energy generation mechanism of the cell
and causes adenosine triphosphate (ATP) to decrease.
An intracellular calcium increase causes proteins and
fats to be damaged by activating proteases and lipases.
Furthermore, ROS also cause damage to DNA, which
results in cell death
17. For this reason, during the
freezing of ram semen, antioxidant substances are
added to semen diluents because it is thought that it
may have positive effects on spermatological
parameters after thawing.
The number of motile spermatozoa is accepted to be an important parameter for fertility 18. Another factor ensuring that the motility in spermatozoa occurs properly is cyclic adenosine monophosphate (cAMP). The increase in cAMP concentration increases the protein kinase activation, so the axoneme protein becomes phosphorylated and causes an increase in the motility of spermatozoon and hyper activation 19. In this study, it was observed that the group containing taurine achieved a significant advantage (P<0.05) in terms of subjective motility after thawing compared to both the control and the other groups. The following research studies have different motility results than those obtained from the antioxidant groups in this study: the motility ratio that Başpınar et al. 20 obtained from the group containing alpha lipoic acid 1 mM of which was added to a tris-based Merino ram semen diluent; the motility ratio that Bucak et al. 21 obtained from 100 mM trehalose added to a ram semen diluent after thawing; the motility ratio that Anghel et al. 22 obtained from the group containing 1 mM vitamin E which was added to a ram semen diluent. The CASA that resulted from the need for an objective, accurate, reliable and repeatable spermatozoon analysis is commonly used for this purpose. The CASA system was developed to monitor the movements of spermatozoa under a photographic microscope many times 23. The fact that some of the values reported in relation to CASA parameters in this study paralleled those from other studies 24,25 indicates that results across studies may be consistent. Furthermore, they are higher than the findings of some researchers 26,27. It is thought that the differences observed in subjective motility and CASA parameters after thawing may be caused by the diluents used in the freezing process, the amounts and ratios of the components contained in the diluents, the changes in thawing time and temperature and the installation and software settings of the device on which the analysis was performed.
The morphological examination of the spermatozoon is important in terms of the abnormal spermatozoa having a low fertilization capacity and some hereditary disorders. There is a close relationship between the localization, variety and quantity of morphological disorders and fertility. In morphological examinations, the acrosome has a particular importance since it plays a critical role in fertilization 18. In the present study, while the findings obtained with regard to the abnormal spermatozoon ratio were found to be lower than the values of some researchers 21,22, it was determined that the values obtained with regard to the acrosome ratio were close to the values reported by Uysal et al. 28 The differences observed in the abnormal spermatozoon and acrosome ratios obtained after the thawing process could be due to genotypic changes in rams, as well as factors such as breed, season and semen collection method.
Membrane integrity is of vital importance not only for spermatozoa metabolism but also for capacitation, acrosome reaction and the realization of the adsorption of sperm to the oocyte surface. For this reason, the HE- test, in which the osmotic tolerance and dead-living examination are combined, is one of the potential sperm function tests used in estimating fertility in humans and other mammals 29. According to the findings of the HOS test and the modified HE-test in which the dead-living spermatozoon ratio was determined in the study, the increases in the taurine, trehalose and lipoic acid groups were found to be statistically significant (P<0.05) in terms of the H+/E- value compared to the control group. This is the first study that evaluates the combination of osmotic tolerance and dead-living examination in cryopreserved ram semen.
Nowadays, the determination of spermatological characteristics, as well as spermatozoon DNA damage, is important for the estimation of male fertility and the sustainability of herd fertility. The comet assay has become a simple, versatile, fast, visual, sensitive and widespread test in which the cells can be evaluated individually 30. Since increased DNA damage is associated with infertility, defective embryonic development, implantation defects, and recurrent abortions, the determination of DNA damage at different levels between the spermatozoa of fertile and infertile males brings the usability of spermatozoon DNA damage as a marker in the evaluation of male fertility potential to the forefront. In particular, the high level of DNA damage in human spermatozoa is associated with infertility 31. The fact that DNA damage obtained from the control and antioxidant groups after thawing parallels the values of other researchers 32,33 indicates that this study is consistent with similar studies. Furthermore, DNA damage was found to be lower when compared to the studies of some researchers 34,35 and higher when compared to others 36,37. Factors, such as breed, semen collection method, the diluents used in the freezing process and the amounts and ratios of the components contained in the diluents, can be considered among the reasons for differences observed in DNA damage after thawing. Factors, such as the difference of antioxidants added to semen diluents and the difference of techniques used in the dilution and freezing of semen may also play a role.
SOD, CAT, and GSH-Px are biological enzymatic antioxidants found in large quantities in seminal plasma. These enzymatic antioxidants protect the motility against LPO by converting superoxide and peroxide radicals into oxygen and water. Moreover, MDA formed as a result of LPO shows the degree of the membrane damage of the spermatozoon 5. According to the findings obtained with regard to oxidative stress parameters in the study, differences were observed between the groups in terms of the relevant parameters. In particular, the decrease in the groups containing lipoic acid and trolox was found to be significant (P<0.05) in terms of MDA compared to the control group. The MDA data obtained in this study are consistent with some studies 20,21. The highest value in terms of GSH was obtained in the control group and in the group containing taurine, and the differences between them and the other trehalose, lipoic acid and trolox groups were found to be statistically significant (P<0.05). The findings of the present study in terms of GSH is parallel the findings of some researchers 20,21. These differences in oxidative stress parameters may have resulted from the factors, such as the differences in the techniques used in the dilution and freezing of semen, and the method used for the analysis of the antioxidant and doses used.
As a result, it was concluded that taurine, trehalose, lipoic acid and trolox used as antioxidants in the freezing of ram semen had different effectiveness; moreover, the lipoic acid achieved an advantage compared to the others because of the oxidative stress and its DNA damage-reducing effect.