Advances in cryopreservation of stallion semen in modified INRA82.

The research article is about an experiment that investigated different methods of improving the post-thaw motility and fertility rates of cryopreserved stallion semen by modifying the INRA82 semen extender.

Research Methods and Approach

• The researchers used a modified INRA82 extender in the cryopreservation process of stallion semen. INRA82 is a commercially available extender containing antibiotics and buffering substances, which is used for fresh and frozen semen preservation. As part of the modification, 2% egg yolk (E1) was added to the INRA82 extender at a temperature of 37 degrees C.
• The semen was then either cooled to 4 degrees C before or after a process of centrifugation and further dilution in E1 with 2.5% glycerol (E2).
• Once cooled, the semen was transferred to 0.5-ml straws and frozen. These straws were thawed at 37 degrees C for 30 seconds prior to performance tests.
• The researchers tested the motility of the semen after thawing; this measurement is referred to as post-thaw motility. Only the samples above 35% post-thaw motility were used for fertility trials.
• Several changes were made during these experimental processes, such as varying the temperature at which the first dilution was performed or coating the spermatozoa with different concentrations of Concanavalin A, to study their impacts on post-thaw motility and fertility.

Key Findings

• The selection of first three jets of ejaculate (as opposed to the whole ejaculate) prior to centrifugation, using of the extender E2 for first dilution instead of E1, and coating the spermatozoa with 0.5 to 8mM of Concanavalin A had no significant impact on post-thaw motility.
• Varying the amount of egg yolk in the E2 extender from 2 to 4% or different glycerol concentrations (range: 1.7-3.7%) also did not significantly influence post-thaw motility.
• Adding 50mM of Glutamine in E2 did result in a slight improvement in post-thaw motility but not in fertility.
• Thawing at 75 degrees C for 10 seconds instead of the usual 37 degrees C had a noticeable but not significant increase in post-thaw motility, but no effect on per-cycle fertility.
• When post-thaw dilution was performed using a fixed molarity multi-step system (25 mOsm per step) from various osmolarities (900-690 mOsm) to 365 mOsm, motility was observed to be unaffected compared with dilution in one step.

Overall Conclusion

The results of this research suggest that most attempted changes in the cryopreservation process do not significantly impact stallion semen post-thaw motility or fertility. Exceptions to this were minimal adjustments to the temperature at which thawing takes place and the inclusion of Glutamine in the E2 extender for minor improvements in post-thaw motility. However, these modifications didn’t significantly influence fertility rates. The authors concluded that further research is required to optimize processes and additives to improve cryopreservation of stallion semen.