Kaempferol as an Alternative Cryosupplement for Bovine Spermatozoa: Cytoprotective and Membrane-Stabilizing Effects

Abstract

Kaempferol (KAE) is a natural flavonoid with powerful reactive oxygen species (ROS) scavenging properties and beneficial effects on ex vivo sperm functionality. In this paper, we studied the ability of KAE to prevent or ameliorate structural, functional or oxidative damage to frozen-thawed bovine spermatozoa. The analysis focused on conventional sperm quality characteristics prior to or following thermoresistance tests, namely the oxidative profile of semen alongside sperm capacitation patterns, and the levels of key proteins involved in capacitation signaling. Semen samples obtained from 30 stud bulls were frozen in the presence of 12.5, 25 or 50 μM KAE and compared to native ejaculates (negative control-Ctrl_N) as well as semen samples cryopreserved in the absence of KAE (positive control-Ctrl_C). A significant post-thermoresistance test maintenance of the sperm motility (p < 0.001), membrane (p < 0.001) and acrosome integrity (p < 0.001), mitochondrial activity (p < 0.001) and DNA integrity (p < 0.001) was observed following supplementation with all KAE doses in comparison to Ctrl_C. Experimental groups supplemented with all KAE doses presented a significantly lower proportion of prematurely capacitated spermatozoa (p < 0.001) when compared with Ctrl_C. A significant decrease in the levels of the superoxide radical was recorded following administration of 12.5 (p < 0.05) and 25 μM KAE (p < 0.01). At the same time, supplementation with 25 μM KAE in the cryopreservation medium led to a significant stabilization of the activity of Mg²⁺-ATPase (p < 0.05) and Na⁺/K⁺-ATPase (p < 0.0001) in comparison to Ctrl_C. Western blot analysis revealed that supplementation with 25 μM KAE in the cryopreservation medium prevented the loss of the protein kinase A (PKA) and protein kinase C (PKC), which are intricately involved in the process of sperm activation. In conclusion, we may speculate that KAE is particularly efficient in the protection of sperm metabolism during the cryopreservation process through its ability to promote energy synthesis while quenching excessive ROS and to protect enzymes involved in the process of sperm capacitation and hyperactivation. These properties may provide supplementary protection to spermatozoa undergoing the freeze-thaw process.

Keywords: Western blot; bulls; cryocapacitation; kaempferol; reactive oxygen species; sperm cryopreservation.

Figure 1

Pre- and post-thermoresistance test motility (a), membrane integrity (b) and acrosome integrity (c) of bovine spermatozoa (n = 30) in fresh state (negative control; Ctrl_N) and cryopreserved in the absence (positive control; Ctrl_C) or presence of different kaempferol (KAE) concentrations. Each experiment was carried out in triplicate. Mean ± S.D. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 2

Pre- and post-thermoresistance test mitochondrial activity (a) and DNA fragmentation (b) of bovine spermatozoa (n = 30) in fresh state (negative control; Ctrl_N) and cryopreserved in the absence (positive control; Ctrl_C) or presence of different kaempferol (KAE) concentrations. Each experiment was carried out in triplicate. Mean ± S.D. ** p < 0.01; **** p < 0.0001.

Figure 3

Pre- and post-thermoresistance test proportion of non-capacitated (a), capacitated (b) and acrosome-reacted (c) bovine spermatozoa (n = 30) in fresh state (negative control; Ctrl_N) and cryopreserved in the absence (positive control; Ctrl_C) or presence of different kaempferol (KAE) concentrations. Each experiment was carried out in triplicate. Mean ± S.D. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 4

Activity of Ca²⁺-ATP-ase (a), Mg²⁺-ATP-ase (b) and Na⁺/K⁺-ATP-ase (c) of bovine spermatozoa (n = 30) in fresh state (negative control; Ctrl_N) and cryopreserved in the absence (positive control; Ctrl_C) or presence of different kaempferol (KAE) concentrations. Each experiment was carried out in triplicate. Mean ± S.D. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 5

Reactive oxygen species (ROS) (a), superoxide (b), hydrogen peroxide (c) and hydroxyl radical (d) production by bovine spermatozoa (n = 30) in fresh state (negative control; Ctrl_N) and cryopreserved in the absence (positive control; Ctrl_C) or presence of different kaempferol (KAE) concentrations. Each experiment was carried out in triplicate. Mean ± S.D. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 6

Protein amounts 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 β-Actin (housekeeping protein) in bovine spermatozoa in fresh state and cryopreserved in the absence or presence of different kaempferol (KAE) concentrations, as determined by Western blotting. Original photos of the gels, membranes and blots are available as Supplementary Materials. Created with (Supplementary: Confirmation of Publication and Licensing Rights) BioRender.com (accessed on 23 December 2023).

Figure 7

Graphical representation of the relative quantification of the CatSper1 (a), CatSper2 (b), NBC (c), PKA (d) and PKC (e) protein in bovine spermatozoa (n = 30) in fresh state (negative control; Ctrl_N) and cryopreserved in the absence (positive control; Ctrl_C) or presence of different kaempferol (KAE) concentrations. Each experiment was carried out in triplicate. Mean ± S.D. * p < 0.05; ** p < 0.01.