152 EVALUATION OF POMEGRANATE (PUNICA GRANATUM) AS REPLICATION INHIBITOR OF BoHV-1 COLORADO STRAIN DURING IN VITRO MATURATION OF INFECTED BOVINE OOCYTES

Abstract

The widespread use of biotechnologies in animal breeding in Brazil has enabled greater control over the transmission of pathogens. Nevertheless, disease transmission continues to be a significant concern and justifies the search for better control agents. The objective of this study was to evaluate whether the detrimental effects of the experimental infection of oocytes with BoHV-1 (Colorado strain, 10⁸ TCID 50 mL^–1) during in vitro maturation (24 h) could be reduced by the viral inhibitor ethanolic extract of Punica granatum (PG). Cattle ovaries were obtained from a local slaughterhouse (unknown breed), and transported to the laboratory. Cumulus–oocyte complexes (COC) were aspirated from follicles and allocated into four groups, which were exposed to: 10 µL of sterile physiological solution (G1 [control], n = 125); 10 µL of PG extract in 0.01% sterile saline solution (G2, n = 149); 10 µL of BoHV-1 virus (G3, n = 151); or 10 µL of PG extract in 0.01% and 10 µL of BoHV-1 virus (G4, n = 144). All groups were matured in 100 mL of IVM for 24 h at 37.5°C, 5% CO₂ in air. After in vitro maturation, we evaluated COC expansion and presence of a polar body by optical microscope, and viral replication by titration (Reed and Muench test) after 72 h co-culture with Madin-Darby bovine kidney (MDBK) cells. Differences among groups in maturation rates were compared by chi-square test, and in titration by t-test. The G1, G2, and G4 showed steady expansion of the cumulus cells and ooplasm with uniform appearance. The G3 did not show expansion of the cumulus cells and ooplasma showed degenerative appearance. The maturation rates were as follows: G1 52% (65/125); G2 45.63% (68/149); G3 27.81% (42/151); G4 41.66% (60/144). We used the χ² test (P ≤ 0.05) for the rate of maturation and the t-test (P ≤ 0.05) for the titration data. A reduction (P ≤ 0.05) in maturation rate was observed in G3, when compared to the G1 [control]. Oocytes exposed to BoHV-1 virus and matured in the presence of PG extract (G4) had lower (P ≤ 0.05) rates of viral replication than those matured in the absence of PG (G3). These results support the conclusion that PG extract reduces the viral rate of replication without interfering in oocyte maturation, and may be an alternative to sanitary control protocols.