Cryoprotective Potential of Theobromine in the Improvement of the Post-Thaw Quality of Bovine Spermatozoa

Abstract

Theobromine (TBR) is a methylxanthine known for its bronchodilatory and stimulatory effects. This research evaluated the vitality, capacitation patterns, oxidative characteristics, microbial profile and expression of capacitation-associated proteins (CatSper1/2, sodium bicarbonate cotransporter [NBC], protein kinases A [PKA] and C [PKC] and adenylate cyclase 10 [ADCY10]) in cryopreserved bovine spermatozoa (n = 30) in the absence (cryopreserved control [Ctrl_C]) or presence of different TBR concentrations (12.5, 25, and 50 µM) in egg yolk extender. Fresh ejaculate served as a negative control (Ctrl_N). Significant post-thaw maintenance of the sperm motility, membrane and DNA integrity and mitochondrial activity (p < 0.001) were recorded following the administration of 25 μM and 50 μM TBR, then compared to Ctrl_C. All groups supplemented with TBR exhibited a significantly lower percentage of prematurely capacitated spermatozoa (p < 0.001) than Ctrl_C. Significantly decreased levels of global reactive oxygen species (ROS), hydrogen peroxide and hydroxyl radicals were observed in the presence of 25 μM and 50 μM TBR (p < 0.01). Western blot analysis revealed that supplementation with 50 μM TBR significantly prevented the loss of NBC and ADCY10 (p < 0.01), while all TBR doses stabilized the levels of PKC (p < 0.05 at 50 μM TBR; p < 0.001 at 12.5 μM and 25 μM TBR). In summary, we suggest that TBR is effective in protecting the spermatozoa during the cryopreservation process through its potential to stimulate energy synthesis while preventing ROS overproduction and the loss of proteins involved in the sperm activation process.

Keywords: bacteria; bull; cryocapacitation; cryopreservation; methylxanthines; protein kinases; semen.

Figure 1

Protein levels of the cation channels of sperm isoforms 1 and 2 (CatSper1 and CatSper2), sodium bicarbonate cotransporter (NBC), protein kinase A (PKA), protein kinase C (PKC) and adenylyl cyclase 10 (ADCY10) in bovine spermatozoa in fresh state and cryopreserved in the absence or presence of selected theobromine (TBR) doses, assessed by Western blotting. Original photos of the gels and blots are available as Supplementary Materials. Created with BioRender.com (accessed on 27 August 2024).

Figure 2

Graphical representation of the relative quantification of the CatSper1 (a), CatSper2 (b), NBC (c), PKA (d), PKC (e) and ADCY 10 (f) proteins in bovine spermatozoa (n = 30) in fresh state (native control (Ctrl_N)) and cryopreserved in the absence (cryopreserved control (Ctrl_C)) or presence of selected theobromine (TBR) doses. Mean ± S.D. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.