INTRODUCTION

Seminal plasma is a very important part of the ejaculate and it is involved in many sperm functions, especially in the metabolism and in events preceding fertilization. But nevertheless, the physiological role of seminal plasma and its components is still not fully understood (1). Also, the effect of seminal plasma on sperm during storage, freezing or cooling is not completely clear.

So far, not many studies about the components of seminal plasma and the freezability of the ejaculate have been conducted. Until now, studies in horses have not shown that it is possible to predict the freezability of semen based on the concentration of some enzymes, microelements (2), total protein or composition of the protein spectrum (3).

However, questionable or different information is found in certain studies or in studies on other animal species (4).

MATERIAL AND METHODS

RESULTS

Average values of basic parameters of ejaculates in both groups are shown in Table 1. The results of the measured selected parameters, which were monitored in the seminal plasma, are arranged in Table 2. Two parameters which were monitored, but not shown in the table, are cholesterol and vitamin A. In all samples the level of these parameters was zero.

Table 1. Average values of basic parameters of ejaculates in groups A and B (Motility II: motility after thawing)

Table 2. Average values of selected biochemical parameters of ejaculates in groups A and B

DISCUSSION

The results of some studies show that seminal plasma negatively affects sperm during short term and also long term storage (5-7). Some other studies denote that in humans (8), and also in stallions (9-11) the presence or adding specific amounts of seminal plasma before storage seems to improve sperm motility during cooled storage and also after thawing. However, the effect of seminal plasma differs significantly between individual stallions (12, 13).

In this study we tried to find differences in the composition of seminal plasma between „good” and „poor” freezable ejaculates in stallions. The only monitored parameter: vitamin E showed statistically significant differences (p≤0,05) between the two groups listed above. It is interesting that higher levels of vitamin E were found in the group of „poor” freezable ejaculates (mean 68 μmol/l), while in the group of „good” freezable ejaculates the mean value of vitamin E was only 15 μmol/l. During semen cryopreservation in humans, the adding of vitamin E to extender in a dose 200 μmol has positive effects on sperm motility in samples from fertile and also subfertile men. When vitamin E in a dose 100 μmol was added, no positive effect on sperm motility was observed (14). Similar observations were made in boars (15), but adding vitamin E to the extender had no effect on sperm motility. When levels of vitamin E were measured in the fresh ejaculate of men (16), no correlation between vitamin E (α-tocopherol) and sperm motility was found. It is a question if the difference between concentrations of vitamin E in our two groups of ejaculates is not just an accidental finding because of the very low concentration of this vitamin in stallion seminal plasma or if sperm motility after freezing and thawing is really influenced by vitamin E. This vitamin is well known as an antioxidant. It is possible that lower use of vitamin E in the antioxidant processes in „poor” freezable ejaculates is the reason for its higher level in this group of ejaculates. However, more samples and further studies are necessary for confirmation or refutation of the results of our study.

The antioxidant role of vitamin E and selenium is very closely related. In the study of Mahmoud et al. (17) it was demonstrated that supplementation of selenium and vitamin E in parenteral form in rams has a positive effect on the quality and quantity of semen. Similar observations were made in stallions (18). The authors proved that oral supplementation with selenium, vitamin E and zinc in stallions leads to the improvement of some quality parameters in ejaculate, especially the improvement of sperm motility. However, the results of another study did not show any improvement of sperm motility in fresh ejaculate and in ejaculate after 24 hours of storage at 5°C when stallions and pony stallions were supplemented with a combination of vitamin E, vitamin C, L-carnitine and folic acid (19). Over the last few years there has been a lot of work on antioxidants, fat acid or commercially available feed additive dietary supplementation, but the results are not very conclusive (20-22). Unfortunately, in none of the studies the levels of supplemented substances were not monitored in seminal plasma.

In this study not only vitamin E was determined, but also selenium. No statistically significant difference was found in the concentration of selenium. From the observations of Bertelsmann et al. (23) it is obvious that in stallions the level of selenium in the blood does not corresponded with the level of selenium in seminal plasma, but the concentration of selenium positively correlated with sperm motility in fresh ejaculate.

In this study, no correlation between the monitored elements was found. The monitored elements were Zn, Cu, Mg, Ca and P. This concurs with the work of Barrier-Battut et al. (2). In humans, a strong correlation between zinc concentration in seminal plasma and sperm motility exists (24, 25), but correlations with other elements has not been confirmed (26). From this view, a measurement of seminal plasma zinc concentration in fertile and subfertile stallions would be interesting.

Concentrations of Na, K and Cl did not significantly differ between both observed groups. The role of these ions during sperm cooling and freezing, as well as the influence on sperm motility before and after storage is still not very clear. There are some studies about these ions in seminal plasma, but the results differ. Also, connection or correlation with semen quality or freezability is still not clear enough (1, 27-30).

The total protein was higher in the group of „poor” freezable ejaculates, but the difference was not statistically significant. It is known that protein concentration is lower in pre-spermatic fraction of ejaculate and the highest protein concentration is in the first phase of spermatic fraction in stallions (31). The total protein seems not to be a very important parameter in stallion ejaculate, but further research of single proteins in seminal plasma could give us some valuable information (1).

Also enzyme concentrations which were monitored in this study are not useful for ejaculate freezability predictions. It has been seen that gama-glutamyl-transferase (GMT) has an important role in sperm protection against oxidative stress, so it was expected that this enzyme could be a good marker for semen freezability (32, 33). Pesch et al. (29) and Kareskoski and Katila (1) found a correlation between GMT concentrations in seminal plasma and sperm motility. In our study, the concentration of this enzyme was higher in the group of „good” freezable ejaculates, but without statistical significance.

A very strong positive correlation between the concentration of lactate-dehydrogenase (LDH) and sperm motility was found (29), but in our study LDH concentrations were almost the same in both groups of ejaculates. Another difference between the „good” and „poor” freezable group was in the concentration of alkaline phosphatase (ALP) but without statistical significance.

Concentrations of ALP were slightly higher in the group of „good” freezable ejaculates. In stallions, as well as in dogs, ALP is an indicator of ejaculation (34, 35). In the study by Kareskoski et al. (30) there was a clear difference between ALP concentrations in seminal plasma in fertile and subfertile stallions. The same result was found in dogs, where there was also a correlation between the concentration of ALP and ejaculate freezability (36). Average values of other monitored enzymes: aspartate-amino-transferase (AST), creatine kinase (CK) and alanine amino-transferase (ALT) was the same in both groups or the difference was minimal.

Creatinine and urea are well known markers for the detection of urine in ejaculate. In both groups, levels of creatinine and urea were almost the same. In all of the samples, the border for urospermia was not exceeded.

Evaluation of the effect of seminal plasma components on sperm and the freezability of ejaculate is complicated. Only little information is available about some of the components, while concerning other components information is not complete, clear or uniform. So, in this part of the research there is still a lot of work and verification to be done.