Impact of tryptophan and trehalose on post-thaw sperm quality and apoptotic gene expression in Wistar albino rats.

Background: Cryopreservation of rat sperm cells and development of new diluents are very important in biotechnology.

Methods: In the current study, 25 mM tryptophan and 100 mM trehalose were added separately to the cryopreservation medium containing 8% lactose monohydrate, 23% egg yolk, and 10% tris aminomethane to evaluate the cryopreservation capability of rat sperms. After freeze-thawing, motility, viable spermatozoon ratio, plasma membrane integrity, abnormal acrosome ratio, and apoptotic cell ratio and levels of Bax, Bcl-2, and Caspase-3 mRNA involved in the apoptosis pathway were evaluated.

Results: The highest viability ratio was detected in the trehalose group (26.25 ± 3.10), which was significantly higher than that in the control group (19.62 ± 1.51) (p<0.05). When compared to the tryptophan group (11.87 ± 3.07), plasma membrane integrity was statistically higher in the trehalose group (20.25 ± 3.65) (p<0.05). The rate of apoptotic cells was lower in the trehalose group than in the control group. Then, the effect of tryptophan and trehalose, which were added to the sperm diluent, on the expression levels of genes in the apoptosis pathway was evaluated through the RT-qPCR technique. The expression of the anti-apoptotic gene Bcl-2 increased by 25 mM tryptophan and 100 mM trehalose, while the expression of the pro-apoptotic genes Bax and Caspase-3 decreased (p<0.01).

Conclusions: As a result, it was determined that trehalose, a isaccharide sugar, added to the rat sperm diluent provided more effective protection against cryodamage in the cryopreservation process than tryptophan, an aromatic amino acid, according to the parameters evaluated. From a molecular biotechnology perspective, these findings lay a valuable foundation for research on the development of next generation media for sperm cryopreservation.