The effects of trehalose on osmotic tolerance, membrane integrity and quality of equine sperm during cold storage and cryopreservation

Abstract

This study aimed to investigate the protective effects of trehalose on osmotic tolerance and the quality of equine sperm before and after freezing and thawing. Equine ejaculated semen was collected from six stallions (3 ejaculates per stallion; >50% motility and >70% normal morphology). In the present study, the osmotic tolerance limit of equine sperm to osmotic changes was firstly verified, and followed with the protective effects of trehalose on osmotic tolerance of equine sperm. In the study, sperm was exposed to Tyrode's albumin lactate pyruvate (TALP) medium at different osmolalities: isosmolality (300 mOsm/kg; control) and anisosmolality (150, 450, 600 and 750 mOsm/kg) and was incubated at 37oC for 10 min. Sperm quality in terms of motility, viability and membrane functionality were evaluated. Results demonstrated that the average of motility was significantly lower in hypo- and hyperosmolality (P<0.05) when compared to isosmolality. The average of viability and membrane functionality were lower in hyperosmolality; however, these were not significantly different in hyposmolality (P>0.05). Moreover, these parameters worsen when the osmolality increased. This data indicated that equine sperm responded to osmotic changes and had limited osmotic tolerance. Co-treatment with 100 mM trehalose revealed that sperm motility, viability and membrane functionality significantly increased when compared to those in the absence of trehalose (P<0.05); except motility in 650 and 750 mOsm/kg (P>0.05). In addition, pre-treatment with 100 mM trehalose, where sperm was centrifuged to remove trehalose and challenged with different osmolalities, increased the sperm quality in term of motility, viability and membrane functionality (P<0.05). Therefore, these results demonstrated that trehalose enhanced osmotic tolerance of equine sperm. In the present study, the protective effects of trehalose on equine sperm before and after cryopreservation were further confirmed. In the first part of the cooling process, equine sperm was pre-equilibrated with isosmotic TALP in the absence (control) and the presence of 100 mM trehalose and incubated at 37oC for 10 min, cooled down to 4oC and maintained for 10 min and 60 min. Compared to the control (no trehalose), trehalose increased sperm quality when maintained in 4oC for 10 min (P<0.05). However, the sperm quality (i.e motility, viability and membrane functionality) were not significantly changed when maintained for 60 min (P>0.05). The second part, during the cryopreservation process, the standard extender were used to cryopreserve sperm in the absence (control) or the presence of 100 mM trehalose. Frozen sperm was thawed at 37oC for 30 second and post-thawed sperm quality was evaluated at 10 min, 2, 4 and 6 h. Results represented that equine sperm quality including motility, viability and membrane functionality had better improved in extender containing trehalose than those without trehalose supplementation (P<0.05) up to 6 hours post-thawing. In conclusion, trehalose enhanced osmotic tolerance of equine sperm and improved equine sperm quality after cryopreservation.