PKA and NBC expression patterns in cryopreserved bovine spermatozoa after kaempferol treatment

Abstract

Kaempferol (KAE) is a member of the flavonoid family, which exhibits antioxidant properties. The richest sources of KAE are green leafy vegetables and herbs like kale, spinach or dill. The goal of our study was to evaluate a possible cryoprotective effects of KAE through to gene expression of protein kinase A (PKA) and sodium bicarbonate cotransporter (NBC). For the experiments we used semen samples obtained from 12 adult Holstein bulls. Before cryopreservation, all samples were divided equally and supplemented by different concentrations of KAE (0, 12.5, 25 and 50 µmol/L), except for native control. After that, RNA was isolated, and the purity and quality of RNA was verified nanophotometrically at A260/A280 nm. Next step was a transcription of RNA into cDNA, which was used for two-step qPCR while expression of PKA and NBC was quantified under specific cycling conditions. Data was evaluated by the delta delta Ct method and One-way ANOVA using Tukey’s range test. According to expression patterns, it was observed that 25 µmol/L of KAE significantly preserved (P<0.0001) the PKA compared to cryopreserved control without KAE treatment. In the case of NBC, there was slightly increase of expression in the group treated with 50 µmol/L of KAE but without significant changes. In conclusion, KAE treatment exhibit promising cryoprotective properties of PKA and NBC genes especially in higher concentration 25 and 50 µmol/L, which may successfully preserved proper expression after thawing of cryopreserved bovine spermatozoa.