Comparative study of the structural integrity of spermatozoa in epididymal, ejaculated and cryopreserved semen of stallions

Cryopreservation of semen is an important way to preserve genetic resources. It is the most actual in horse breeding than in other livestock industries, as currently in many horse breeds, especially unique domestic breeds, the number is approaching a critical level. For many native breeds due to a specific management, year-round outdoors in herds, it is impossible to obtain sperm for cryopreservation by a traditional method using an artificial vagina, and the only cost-effective way to create cryobanks is getting epididymal semen. The technology for cryopreservation of stallion semen includes some critical steps that are characterized by a decrease in sperm quality. These are procedure for semen donation, dilution, temperature shock during freezing and thawing. For the first time a comparative transmission electron microscopy study of the ultrastructural integrity of spermatozoa was done for ejaculated and epididymal sperm in the same stallions thus avoiding the influence of different individuals on the compared parameter. Structural damage caused by cryoconservation of epididymal and ejaculated sperm was studied. It was found that acrosomes were the most susceptible and undergone the greatest impact. Its predominant pathology is the absence of internal contents (acrosome hypoplasia), resulted in enzyme deficiency. The frequency of this pathology was 12.4 % and 14.0 % for fresh epididymal and ejaculated sperm, respectively, and increased almost twofold after freezing and thawing, reaching 26.5 and 27.4 %, respectively. The rate of spermatozoa with the second most common pathology, acrosome degradation (premature release of sperm enzymes that dissolve the oocyte membrane, resulting in the loss of the ability to fertilize), after cryopreservation increased by 5.9 % (p < 0.05) and 8.9 % (p < 0.01) for epididymal and ejaculated semen, respectively. The nucleus of the sperm is one of the most resistant to cryopreservation among the organelles, though in two stallions we observed changes in the shape of the nucleus and vacuolation of chromatin after cryopreservation with a frequency of 1.6 to 6.1 %. Less than 10 % of the sperm had pathology of mitochondria. The axoneme of sperm is sufficiently resistant to cryopreservation. Outer dense fibers and fibrous membrane are almost not damaged during semen collecting as well as under semen freezing and thawing. Higher rates of ultrastructural integrity found for epididymal semen were not statistically significant. Thus, the sperm collecting results in minimal ultrastructural damage, whereas the main pathology is caused by cryopreservation. The ultrastructural integrity of spermatozoa in epididymal semen allow us to recommend this sperm collecting technique to organize cryobanks in case of impossibility of sperm collecting from stallions in traditional ways, or in need for early castration of stallions, in particular in sporting and productive horse breeding.