172 ANTIVIRAL ACTION OF PROPOLIS AQUEOUS EXTRACT DURING MATURATION OF INFECTED BOVINE OOCYTES

Abstract

The objective of this study was to evaluate the decrease of virus replication in BoHV-1 (Colorado strain, 10^6.5 TCID₅₀/mL) after the treatment using propolis aqueous extract (PAE) during in vitro maturation of infected bovine oocytes (24 h). Cow ovaries were obtained from a local slaughterhouse (Nelore breed) and transported to the laboratory. Cumulus-oocyte complexes (COC) were aspirated from follicles and separated into 4 groups (number of replicates for all groups = 6), which were exposed to 20 μL of sterile physiological solution (SPS), 100 μL of the in vitro maturation (IVM) medium [G₁ (control), n = 609]; 10 μL of BoHV-1 (10^6.5 TCID₅₀/mL) virus, 100 μL IVM medium, and 10 μL of SPS (G₂, n = 786); 10 μL of PAE in 0.001% in SPS, 100 μL IVM medium, and 10 μL of SPS (G₃, n = 819); 10 μL of PAE extract in 0.001% in SPS, 10 μL of BoHV-1 (10^6.5 TCID₅₀/mL) virus and 100 μL of the IVM medium (G₄, n = 734). All groups were kept for 24 h at 38.5°C, 5% CO₂ in air. After the IVM, we analysed COC expansion and the presence of a polar body by optical microscope as well as viral replication by titration (Reed and Muench test) after 72 h of co-culture with Madin-Darby bovine kidney (MDBK) cells. The G₁, G₃, and G₄ showed steady expansion of the cumulus cells and ooplasm with uniform appearance. The G₂ did not have expansion of the cumulus cells. In contrast, the cytoplasm showed degenerative appearance and an absence of maturity in numerous oocytes. The maturation rates were as follows: G₁ = 79% (482/609), G₂ = 51% (407/786), G₃ = 80% (662/819), and G₄ = 76% (565/734). The differences among groups in maturation rates were compared using the chi-squared test (α = 5%) and the average titrations using the Mann–Whitney test (α = 5%). There was a significant difference (P < 0.01) among G₁ and G₂ evincing the interference of the virus maturation. The extract did not affect maturation as there was no difference among G₁ and G₃ (P = 0.43). The main step was finding no significant difference between the groups G₁ and G₄, (P = 17) proving that the extract interferes with viral replication. The titration after co-culturing the oocytes in MDBK demonstrated that G₄ (average titrations = 1.63 × 10³ titration) showed a lower rate of viral replication, the Mann-Whitney test, when compared to group G₂ (average titrations = 6.04 × 10⁷) which has not been subjected to treatment with PAE (P = 0.02). These results indicate that the propolis aqueous extract reduces the rate of viral replication without interfering with the maturation of oocytes and, therefore, it can be a conclusion that the analysis of the action of the molecules of this extract (by proteomics, for example) and future studies should be directed towards identifying the effect of extract on antiviral activity during the assessment of oocyte competence and embryo development.