Magali D'Angelo

Acceptable microorganisms concentration in a semen sample for in vitro embryo production

The aim of this study was to report that the acceptable concentration of microorganisms in a semen sample for insemination may not be safe for an in vitro fertilization procedure. It seems that the semen sample should be completely germ-free, because of the excellent microorganism proliferation condition promoted by the in vitro environment.

Desempenho reprodutivo entre receptoras bovinas não contaminadas e naturalmente infectadas por Neospora caninum, após a transferência de embriões

The use of knowledge in biotechnologies for improving the production of goods and services has advanced significantly. The control of infectious diseases continues to represent the most obstacle to the health of animals. The embryonic loss may be responsible for the increase in economic losses for producers isolated from cattle. Infection with Neospora caninum has emerged as an important reproductive disease and, in several countries, has been diagnosed as the main cause of abortion. The aim of this study was to evaluate the reproductive performance in not contaminated and naturally infected with Neospora caninum bovine receipt after embryos transfer (ET). The choice of animals (n=395) for the experiment was performed by gynecological evaluation, via transrectal palpation. Vaccinations recommended for health protocol followed the ET. Nutritional management was based on the National Research Council (NRC, 1999). The ET of thawed embryos was made after the synchronization of the estrous cycle of the receipts. To evaluate the reproductive performance of transrectal ultrasonography was used. The identification of the positive receipts for Neospora caninum was performed by ELISA and reagents for samples, confirmation by indirect immunofluorescence (IIF). Data were analyzed using Past® and were considered statistically significant (P<0.01). The results show a rate of pregnant (n=191) of 48.35%, followed by an abortion rate at 60 days of gestation (n=14) and 3.5% of abortions in the sixth month of gestation (n=12) of 3.04%. These results become relevant when taking into consideration that infected receipts by the protozoan not showed a high rate of pregnancy loss. The statistical insignificance of the data obtained in this study provides the security suggesting that there is no need to deploy the diagnosis or prior control to neosporosis in receipts able to ET.

Cultivo primário de células epiteliais germinativas do limbo de olhos doadores

OBJETIVO: Padronizar cultivo primario de celulas epiteliais germinativas do limbo de olhos doadores do Banco de Olhos da Santa Casa de Sao Paulo. METODOS: Por meio de biopsias de aproximadamente 4 mm² realizadas na regiao do limbo cirurgico do anel corneo-escleral remanescente de olhos doadores, foram obtidos explantes, que passaram por metodos de dissociacao para obtencao de celulas epiteliais germinativas do limbo, as quais foram semeadas em placas de cultivo celulare encubadasna estufa a 37°C em atmosfera a 5% de CO2. As placas foram observadas em 21 dias, sendo avaliadas a aderencia, a morfologia e a multiplicacao celular. RESULTADOS: Observamos que as celulas no decorrer dos 21 dias multiplicaram-se, passando da forma arredondada inicial ao processo de dissociacao, para a forma poligonal como in vivo, alem de demonstrarem maior aderencia a placa de cultivo. CONCLUSAO: Neste estudo foi possivel demonstrar que o cultivo de celulas epiteliais limbicas in vitro pode ser realizado.

In vitro interaction of bovine herpesvirus 1 with uterine tube epithelial cells and oocytes

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.

Alterações na morfologia e na viabilidade no desenvolvimento de embriões bovinos fecundados in vitro com sêmen contaminado experimentalmente à Escherichia coli produtora da toxina Shiga stx2

The objective of this study was to evaluate by optical microscopy and transmission electron, changes in morphology and viability of the development of bovine embryos, fertilized with semen experimentally contaminated (STEC). Oocytes were aspirated from ovaries of slaughtered cows and the intact zona pellucida were selected and matured. After 20-24 hours of maturation, the oocytes were divided into 2 groups. The first, control group (n = 4l8),fertilized with semen tested and without any type of contaminant and the second, the infected group (n = 415), fertilized with sperm exposed to STEC. Both semen were treated by the technique of discontinuous Percoll gradient. After the period of fertilization, embryos were evaluated for their morphology and viability by optical and electron microscopy. In morphologic evaluation, the oocytes fertilized with contaminated semen showed cytoplasmic shrinkage, gaps in the division, asymmetry of blastomeres, ooplasm grainy, dark brown color, vacuoles formation, degeneration and zona pellucid disruption. These changes were not observed in the control group. The cleavage rate was 70.3 and 52.8%, respectively, for control and infected groups, significant differences (p = 0.0001). After the 5th day of embryonic development, where it was observed 44.7% of morula in the control group, and 22.4% in the contaminated group, showing a significant difference (p = 0.0001). The presence of STEC interferes with the cleavage rate of embryos and also prevents and causes a decline in embryonic development to the morula stage and cause morphological changes during this development.

Interação do Mycoplasma bovigenitalium com células do cumulus in vitro após o período de maturação oocitária bovina

The Mycoplasma is considered cosmopolitan and can be disseminated through international trade of animals, industrialized semen and embryo transfer products. The expansion of cumulus cells is used as a parameter for evaluating cattle oocytes cultivated in vitro, and their morphological changes are representative. The aim of the present study was to evaluate the interaction of Mycoplasma bovigenitalium with primary-culture cumulus cells, after the in vitro maturation period. Cumulus complex oocytes (COCs) obtained through follicular puncture of ovaries from a cattle slaughterhouse were divided into two groups to be matured for 24 hours in the maturation medium (TCM 199 + hormones) in a climate controlled chamber at 38o C, 5% CO2, and 95% humidity. Subsequently, the oocytes were removed from the plates, which remained with only the cumulus cells attached. With the monostratum cell formed, one group was infected with 30 oL (5 x 106 cells/mL) M. bovigenitalium, replicated in modified Hayflick medium at 37o C in a mycroaerofilic chamber, while the other was kept as a control. The results showed that with 24 hours of exposure to the pathogen, the cultures showed a small number of rounded and grainy cells, when compared to the controls. This effect persisted until the 7th day, when a process of cell detachment began. It can be concluded that a mycoplasma contamination may be imperceptible to the manipulations of FIV, because cells infected by this group of bacteria present no cloudiness in the culture field, and when they do not lyse the host cell, they make it more susceptible to environment and other infectious agents.

297 ASSESSMENT OF THE TRANSPORTATION OF BOVINE OOCYTES EXPERIMENTALLY EXPOSED TO BOVINE HERPESVIRUS TYPE 1 (BoHV-1) DURING THE PERIOD OF MATURATION IN IN VITRO OVIDUCTS

The necessity of greater elucidation about oocyte pathogen interactions made us examine the interaction of bovine herpesvirus type 1 (BoHV-1), Los Angeles strain, with oocytes matured in vitro to assess the potential hazard of transmission of such infectious agent through IVF. The process of capacitation and transportation of oocytes through the oviduct depends on the quality of the female gamete. Keeping in mind that the BoHV-1 causes morphophysiological modification in oocytes, the aim of this study was to evaluate, in an experimental model of in vitro oviduct, whether the pathogen interferes in the transportation of oocytes. This was determined using the percentage of oocyte recovery and assessment of the alteration in the morphology of the oviduct lumen. Oviducts and oocytes were collected from bovine ovaries derived from slaughterhouse cows. The oviducts were dissected, washed, and placed in Petri dishes (100 × 20 mm). On the previous portions of the infundibulum, in vitro-matured control oocytes or oocytes exposed to the virus (105.5 TCID50 mL-1) were introduced (20 oocytes per group) and then immersed in 100 mL of TCM-199 medium. Each dish was incubated at 38°C, 5% CO2, and 95% humidity for 24 h. Then, we introduced in the infundibulum, through intramammary infusion probe, 5 mL from a physiologic solution containing 1% of bovine fetal serum. Each oviduct effluent was collected separately and evaluated through a stereoscope for the recovery and counting of oocytes. Immediately after the recovery, both oviduct groups were sectioned longitudinally and observed through an optical microscope (100 ×) for a morphological evaluation of the luminar area. For the control group, the percentage of recovery was 14.9% (65/437), whereas in the infected group, the oocyte recovery was 23.4% (100/428). The statistical analysis was made according to Student’s t-test (P < 0.05), and presented a significant difference in the final results. The previous results show that the group of oocytes that was exposed to the virus presented a higher percentage of recovery compared with the control group. The oviducts that received the exposed oocytes presented areas where it seemed there has been cytopathic effect represented by dark and lumpy sections, in which there were round and degenerating cells mainly located in the oviducts’ peripheral area. The morphophysiological modifications in contaminated oocytes and oviducts probably complicate their transportation and also interferes in the process of their liberation. It is relevant to elucidate the importance of the studies above because of their participation in the recovery of these gametic cells in bovine oviducts, as well as the transmission during IVF processes. Vitrocel/Embriolife.

274 ASSESSMENT OF TRYPSIN AND ANTIBIOTIC TREATMENT EFFECTIVENESS IN IN VITRO-MATURED OOCYTES EXPERIMENTALLY EXPOSED TO LEPTOSPIRA INTERROGANS SEROVAR GRIPPOTYPHOSA

Techniques of production and transfer of embryos is safe as long as it follows the control regulations defined by the manual of the International Embryo Transfer Society (IETS) for treating oocytes with trypsin, antibiotics, and TCM-199 medium. The aim of this work was to evaluate the effectiveness of treatments, established by IETS, in bovine oocytes experimentally exposed to Leptospira interrogans serovar Grippotyphosa and to assist implementation of quality control standards on in vitro embryo production. The oocytes were obtained through follicular puncture of ovaries derived from the slaughterhouse. They were selected and divided into 4 groups: the control group and groups exposed to 5, 10, and 30 μL of an L. interrogans strain at 4.7 × 105 μL-1; and 4 additional groups exposed to the same concentrations of another L. interrogans strain at 6.3 × 105 μL-1, in which the gene for virulence is not expressed. The groups were kept in maturation medium (TCM-199 medium, 0.5 (iLof FSH, 50IU mL-1 hCG, 1μL mL-1 17-βiestradiol) and incubated at 38°C, 5% CO2, and 95% humidity for 24 h. All the groups were separately subjected to the treatments with antibiotic, trypsin, and TCM-199 medium after maturation. The treatment involved 10 drops (each 200 μL), with 8 drops of TCM-199 medium and 2 drops of antibiotic (penicillin 10000 IU mL-1 and streptomycin 10 mg mL-1) or trypsin 0.25%; exposed to trypsin and antibiotic for 120 s. For the sequential washes, all drops contained TCM-199 medium. The analysis for presence of the pathogen by dark-field optical microscopy showed that in the groups exposed to L. interrogans and subjected to antibiotic washes, the effectiveness was 50% (100/200) for the group exposed to 5 μL, 40% (80/200) for that exposed to 10 μL, and 22.5% (45/200) for that exposed to 30 μL. We found the same results after the trypsin washes. After the washings with TCM-199 medium, the groups infected with 5 and 10 μL presented 100% of effectiveness; however, for the group infected with 30 μL, the washings were not effective. For the groups exposed to L. interrogans that did not express virulence, after the washings with antibiotics as well as with trypsin, the results showed no effectiveness in all of them (n = 200). Yet, after washings with TCM-199, the group exposed to 5 μL showed 28.5% (57/200) of effectiveness, whereas in those exposed to 10 and 30 μL, the medium washes were not effective. Complementary studies are being made with ultramicrotome cut and polymerase chain reaction for more reliable conclusions, to confirm the results. With such results, we conclude that the quality control regulations established by IETS for IVP could be reviewed and possibly redefined, because the effectiveness of the treatment may depend not only on the pathogen species, but also its virulence as well as its concentration and the action of the treatments on the type of pathogen. We thank Vitrocel/Embriolife for supporting the laboratory.