The aim of this study was to evaluate the efficiency of trypsin treatment on the inactivation of bovine herpesvirus type 1 (BoHV-1) on in vitro produced by fertilization and artificially infected bovine embryos. Bovine embryos on day 7 were exposed with 10 microl of BoHV-1, Los Angeles strain 10(7.5) TCID. These embryos and control embryos were divided in two groups: submitted to the sequential washes or to the trypsin treatment according to the International Embryo Transfer Society (IETS) guidelines. The embryos and the last washing drop of each group were used as inoculum to infect Madin Darby bovine kidney (MDBK) cells and submitted to nested PCR reaction using the primer that encodes the gene conserved region of virus glycoprotein gB. The data have shown that the control embryos and their last washing drop were negative. The exposed embryos that were treated with trypsin have shown positive results on the n-PCR and MDBK culture, and their last washing drop were negative. Our data have demonstrated that the trypsin treatment was not able to eliminate the BHV-1 of the embryos, suggesting an interaction between virus and embryo.
The aim of this study was to evaluate the capacity of three semen processing techniques, Percoll gradient centrifugation, Swim-up and a combination of Swim-up and Percoll gradient centrifugation, to reduce the viral load of bovine viral diarrhea virus (BVDV) in experimentally infected semen samples. The evaluation was performed using two approaches: first, searching for the presence of virus in the processed samples (via virus titration and RT-PCR) and second, ascertaining the possible interference on in vitro embryo production. The sperm count and DNA integrity (Comet assay) of the processed samples were analyzed (Experiment 1). The amount of virus in the processed samples was determined by titration in cell culture (Experiment 2). The samples processed by Swim up/Percoll gradient centrifugation were utilized for in vitro embryo production, and the embryos produced were tested for BVDV by RT-PCR (Experiment 3). Sperm concentration, Comet assay and embryo production were analyzed by chi-squared tests (P<0.05). There was a significant difference between sperm separation techniques when the sperm count and Comet assay were analyzed. The sperm count obtained from the Swim up/Percoll gradient centrifugation group was lower than that obtained in either of the two other groups (Swim up and Percoll gradient centrifugation), and the Comet assay showed that the combination of the two semen processing techniques (Swim up/Percoll gradient) produced a 1.1% prevalence of Comet level 2, which was not observed in the other groups. The BVDV titer (10(6.68)TCID(50)/mL) added to experimentally infected semen samples decreased after Percoll gradient centrifugation to 10(2.3)-10(1)TCID(50)/mL; for the Swim up group, the titer range was 10(3.3)-10(1.87)TCID(50)/mL, and in the Swim up/Percoll gradient centrifugation group, BVDV was undetectable. The decreases in titer varied from 99.9% in the Swim up-processed group to 100% in the Swim up/Percoll gradient centrifugation group. In vitro embryo production displayed similar blastocyst development rates among all groups, and RT-PCR was negative for the produced embryos. The data showed that the combination of Swim up/Percoll gradient centrifugation promoted the elimination of BVDV from the semen samples without damaging spermatozoa cells and also allowed successful in vitro embryo production free of BVDV. Hence, the risk of BVDV contamination is negligible for the embryo recipient.
The aims of this study were to assess in vitro if bovine oocytes and oviductal epithelial cells from slaughterhouses for in vitro fertilization use may be infected with bovine herpesvirus 1; to analyze whether the treatment with trypsin according to the International Embryo Transfer Society guideline is efficient to inactivate the bovine herpesvirus 1; to morphologically study the virus-oocyte interaction through optical microscopy. In this study, Madin Darby Bovine Kidney (MDBK) cells that were co-cultured with oocytes matured in vitro and exposed to bovine herpesvirus 1 showed a cytopathic effect. The nested polymerase chain reaction for the supernatant was positive for the bovine herpesvirus 1, thus suggesting that the cytopathic effect observed in the MDBK monolayer was seen due to virus replication and not because of any culture toxicity. It was also observed cytopathic effect and positive nested polymerase chain reaction in MDBK cells co-cultured with in vitro maturated oocytes free of virus, but that were co-cultured in uterine epithelial cells pre-infected with bovine herpesvirus 1 and washed or not with trypsin, demonstrating an oocyte contamination by the virus. When trypsin-washing efficacy was evaluated, we could observe that the trypsin treatment was not able to eliminate the bovine herpesvirus 1 of the oocytes, and it was not observed any morphological difference in the infected oocytes.
In vitro embryo production (IVP), as well as having a biotechnical importance, is a valuable tool for studies of gamete and/or embryo interaction with pathogens and xenobiotics. In consequence, it has become an excellent model not only for investigations about sanitary aspects, but also for aspects related to toxic processes. The aim of this study was to evaluate the effect of cytotoxic aqueous extract of Ateleia glazioviana and its interference on the interaction of bovine herpesvirus type 1 (BoHV-1) with bovine oocytes during the In vitro maturation (IVM) period. The statistical analysis of the experiments was made according to Student’s t-test (P < 0.05). The parameters used for this experiment were based on the morphological, physiological, and clastogenic action analysis of the bovine oocytes. The oocytes were collected from ovaries from slaughterhouse and divided into control group (G1, n = 214), a group infected with BoHV-1 (Los Angeles sample 105.5 TCID50 mL-1(G2, n = 210), a group exposed to the extract of A. glazioviana, 0.24 g mL-1; G3, n = 228), and a group simultaneously exposed to the virus and to the extract (G4, n = 210). For IVM, the oocytes were kept in TCM-199 supplemented with hormones and incubated at 38°C, 5%CO2, and 95% humidity for 24 h. The oocytes in G1 showed high expansion of the cumulus cells and ooplasm uniform in appearance; oocytes in G2 showed uniform but moderate expansion of cumulus cells and retraction of ooplasm; the G3 group showed low and irregular expansion with degeneration of cumulus cells and retraction of ooplasm with a granular aspect; and oocytes in G4 showed degeneration of cumulus cells, retracted and granular ooplasm. We observed maturation rates of 81.3% in G1, 31.0% in G2, 5.7% in G3, and 1.4% in G4. As for the clastogenic action analysis, an additional group of oocytes, named in natura (n = 210), was evaluated and presented 41.9% of comets class 0 (zero), 34.8% class I, 12.4% class II, 7.1% class III, and 3.8% class IV G1 (n = 211) presented 6.1% of comets class 0, 47.8% class I, 31.3% class II, 11.0% class III, and 3.8% class IV Oocytes belonging to G3 (217) presented 0.5% of comets class 0, 19.8% class I, 28.1% class II, 34.1% class III, and 17.5% class IV G2 (n = 229) presented 4.4% of comets class 0, 61.2% class I, 26.6% class II, 4.8% class III, and 3.0% class IV Oocytes in G4 (n = 206) presented 3.9% of comets class 0, 26.2% class I, and similar amounts of comets level II (23.8%), III (22.8%), and IV (23.3%). The statistical analysis presented a significant difference in the final results. Such results show the cytotoxic effect of A. glazioviana in bovine oocytes. The simultaneous exposure to the virus and the extract aggravated the effect of the virus, suggesting an increase of the pathogen within the gametic cell. Vitrocel/Embriolife.
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.
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