Effect of Seminal Plasma Protein Fractions on Stallion Sperm Cryopreservation.

Seminal plasma (SP) is the natural environment for spermatozoa and contains a number of components, especially proteins important for successful sperm maturation and fertilization. Nevertheless, in standard frozen stallion insemination doses production, SP is completely removed and is replaced by a semen extender. In the present study, we analyzed the effects of the selected seminal plasma protein groups that might play an important role in reducing the detrimental effects on spermatozoa during the cryopreservation process. SP proteins were separated according to their ability to bind to heparin into heparin-binding (Hep+) and heparin-non-binding (Hep-) fractions. The addition of three concentrations-125, 250, and 500 µg/mL-of each protein fraction was tested. After thawing, the following parameters were assessed: sperm motility (by CASA), plasma membrane integrity (PI staining), and acrosomal membrane integrity (PNA staining) using flow cytometry, and capacitation status (anti-phosphotyrosine antibody) using imaging-based flow cytometry. Our results showed that SP protein fractions had a significant effect on the kinematic parameters of spermatozoa and on a proportion of their subpopulations. The 125 µg/mL of Hep+ protein fraction resulted in increased linearity (LIN) and straightness (STR), moreover, with the highest values of sperm velocities (VAP, VSL), also this group contained the highest proportion of the fast sperm subpopulation. In contrast, the highest percentage of slow subpopulation was in the groups with 500 µg/mL of Hep+ fraction and 250 µg/mL of Hep- fraction. Interestingly, acrosomal membrane integrity was also highest in the groups with Hep+ fraction in concentrations of 125 µg/mL. Our results showed that the addition of protein fractions did not significantly affect the plasma membrane integrity and capacitation status of stallion spermatozoa. Moreover, our results confirmed that the effect of SP proteins on the sperm functionality is concentration-dependent, as has been reported for other species. Our study significantly contributes to the lack of studies dealing with possible use of specific stallion SP fractions in the complex puzzle of the improvement of cryopreservation protocols. It is clear that improvement in this field still needs more outputs from future studies, which should be focused on the effect of individual SP proteins on other sperm functional parameters with further implication on the success of artificial insemination in in vivo conditions.

How to Increase Post-Thaw Semen Quality in Poor Freezing Stallions: Preliminary Results of the Promising Role of Seminal Plasma Added after Thawing.

The aim of this study was to evaluate the effect of the addition of two types of seminal plasma (SP) after thawing on the functional characteristics of frozen-thawed (F-T) spermatozoa of poor freezing stallions during prolonged incubation periods. Seminal plasma from stallions with 35-40% (standard seminal plasma, (S-SP)) and 60-70% (above standard seminal plasma, (A-SP)) progressively motile spermatozoa after thawing was used. The motility, kinematic parameters (Computer Assisted Sperm Analysis), distribution of spermatozoa into subpopulations, integrity (carboxyfluorescein diacetate/propidium iodide staining), and functionality (hypo-osmotic swelling (HOS) test) of the spermatozoa plasma membrane were evaluated after thawing (T0) and after 30 min (T30) of incubation at 37 °C. There was no effect of SP addition on spermatozoa motility, but there was a significant positive effect on the kinematic parameters at T0 and T30. The addition of SP significantly increased the percentage of spermatozoa in the fast subpopulation at T0 as well as at T30. Plasma membrane integrity was not affected by the treatment, but functionality significantly decreased by 5% compared to the control group when samples were incubated for 30 min with A-SP. In conclusion, generally, the post-thaw addition of seminal plasma positively affected the post-thaw quality of semen from poor freezing stallions.

Improvement in Semen Conservation of the Indigenous Czech Endangered Old Kladruber Horse: Special Focus on the Type of Extender and Packaging System.

The aim of our study was to investigate the effect of two freezing extenders and two packaging systems on motility, plasma membrane (PM) integrity, and the apoptotic status of frozen-thawed (F-T) spermatozoa of the endangered Old Kladruber stallions. The collected semen (n = 6 stallions, three collections each) was diluted either with Gent or Lactose-EDTA (Lact) extender. Two aliquots of semen from each collection diluted in this way were prepared and then loaded into 5-mL aluminum tubes or 0.5-mL plastic straws. After thawing and then at 15 minutes intervals within 1 hour, the samples were analyzed for motility (CASA), PM integrity (CFDA/PI), and apoptotic changes of the spermatozoa (Yo-Pro-1/PI). Using Gent, the samples exhibited higher motility, kinematic parameters, higher representation of spermatozoa in medium, and fast subpopulation, as well as more spermatozoa with intact PMs and fewer spermatozoa with apoptotic changes compared with Lact extender (P < .05). Progressive motility and PM integrity was superior when using Gent and 5-mL aluminum tubes as compared with the rest of the combinations (P < .05). Kinematic parameters, percentage of spermatozoa in fast subpopulation and apoptotic status was superior in Gent and 0.5-mL straws as compared with the rest of the combinations (P < .05). Moreover, we revealed that F-T semen reacts diametrically differently when two different extenders and packaging systems are used. The study concludes that the combination of Gent and 0.5-mL straws represent adequate freezing system to maintain the appropriate quality of spermatozoa of this endangered breed.