Hepatitis E in a Portuguese cohort of human immunodeficiency virus positive patients: High seroprevalence but no chronic infections.

Introduction: Hepatitis E virus (HEV) infection causes zoonotic hepatitis in Europe, with a higher risk of complications in immunocompromised hosts. HEV natural history in human immunodeficiency virus (HIV) positive patients is not fully understood, and its prevalence is unknown. Objectives: To study the seroprevalence of HEV and prevalence of chronic HEV in HIV-positive patients from Porto, Portugal. Methods: We randomly selected patients from the cohort of HIV-positive patients followed in our hospital. We performed an enzyme-linked immunosorbent assay to search for immunoglobulin G for HEV. When the absorbance/cut-off was inferior to 3.5, the test was repeated, and a confirmatory test executed in that sample. For reactive tests and for immunosuppressed patients (CD4 count < 200/mm3) with nonreactive test, a polymerase chain reaction (PCR) test was also performed. Results: We included 299 patients. The mean age was 48 and 75.3% were men. Regarding HIV infection, the median follow-up time was 10 years, the acquisition was mainly heterosexual contact, and 94% were on antiretroviral therapy. Seventy-six patients (25.4%) had reactive immunoglobulin G (IgG) hepatitis E serology. Patients with a reactive test were older (statistically significant difference). Otherwise, there was no difference between groups concerning birthplace, rural residence, chronic viral hepatitis coinfection, or cirrhosis. Nadir and actual TCD4+ lymphocyte counts did not differ significantly from patients with HEV reactive and nonreactive serology. Gamma-glutamyl-transferase (GGT) was higher in patients with reactive IgG HEV. All serum HEV PCR tests were negative. Conclusions: Seroprevalence of HEV was 25.4% in HIV-positive patients. Older age and higher GGT correlated to HEV reactive IgG test. No cases of current hepatitis E were found.

Effects of paternal diet and antioxidant addition to the semen extender on bovine semen characteristics and on the phenotype of the resulting embryo.

The aim of this study was to examine the effects of long-term dietary supplementation of young Nellore bulls with rumen-protected polyunsaturated fatty acids (PUFAs) and of the inclusion of catalase in the semen extender on semen quality, in vitro sperm fertilizing ability, and intracytoplasmic lipid content in the resulting embryos. Twelve Nellore bulls were supplemented with rumen-protected PUFAs or with a basal diet from 14 to 24 months of age. The semen was collected at the end of supplementation. For cryopreservation, the ejaculate was divided into two equal volumes and catalase was added to the extender in one of the fractions. Thus, the experimental design consisted of a 2 × 2 factorial scheme with two diets (control and PUFA) and two extenders (Cat+ and Cat-). Total motility and the percentage of rapid cells in fresh semen were negatively affected by dietary supplementation with PUFAs (P < 0.05), but these effects did not persist after freezing. The frozen/thawed semen of animals fed PUFAs exhibited an increase in the percentages of damaged plasma and acrosomal membranes, as well as an increase in the proportion of lipids ions at m/z 578 and m/z 757 detected by MALDI-MS. Nevertheless, there was no effect of the treatments on in vitro embryo development. However, embryos derived from bulls supplemented with PUFAs exhibited higher lipid accumulation compared to control (P < 0.05). In conclusion, PUFA supplementation promoted worsening of semen quality without affecting the in vitro sperm fertilizing ability; however, the paternal diet affected the intracytoplasmic lipid content in the resulting embryos.

Infectious Risk Mitigation in Patients with Multiple Sclerosis under Disease-Modifying Therapies - the Experience of a Collaborative Neurology-Infectious Diseases Approach.

BACKGROUND: Multiple sclerosis treatment has changed in the last years with the emergence of new disease-modifying therapies (DMTs). Despite a better efficacy profile, these drugs raise concerns about infectious risk, which needs to be mitigated. OBJECTIVE: To analyze the results of a systematic collaborative approach between Neurology and Infectious Diseases (ID) Departments in the management of infectious risk and complications in MS patients treated with DMT. METHODS: Retrospective collection of MS patients' demographic and clinical data from clinical records of MS and ID outpatient clinics (2011-2017). RESULTS: We included 149 patients: most had evidence of previous contact with Herpesviridae, and half of them were not immune to hepatitis A and B viruses (HAV and HBV). Vaccines for HAV, HBV, and Streptococcus pneumoniae were administered in 91%, 78%, and 88% of non-immune patients, respectively. JC virus serology monitoring prevented natalizumab (NTZ) initiation or prompted its switch in 34/122 patients. Forty patients had latent tuberculosis, in which 88% were treated. Infectious events occurred in 33 patients, mostly mild urinary, respiratory, and herpes virus group infections. Only three patients required inpatient care. CONCLUSION: Facing the expansion of the new DMT, we highlight the benefits of an interdisciplinary approach for safer use of the chosen treatment.

Telehealth to support referral management in a universal health system: a before-and-after study.

BACKGROUND: Management of patient flow within a healthcare network, allowing equitable and qualified access to healthcare, is a major challenge for universal health systems. Implementation of telehealth strategies to support referral management has been shown to increase primary care resolution and to promote coordination of care. The objective of this study was to assess the impact of telehealth strategies on waiting lists and waiting times for specialized care in Brazil. METHODS: Before-and-after study with measures obtained between January 2019 and February 2020. Baseline measurements of waiting lists were obtained immediately before the implementation of a remotely operated referral management system. Post-interventional measurements were obtained monthly, up to six months after the beginning of operation. Data was extracted from the database of the project. General linear models were applied to assess interaction of locality and time over number of cases on waiting lists and waiting times. RESULTS: At baseline, the median number of cases on waiting lists ranged from 2961 to 12,305 cases. Reductions of the number of cases on waiting lists after six months of operation were observed in all localities. The magnitude of the reduction ranged from 54.67 to 88.97 %. Interaction of time measurements was statistically significant from the second month onward. Median waiting times ranged from 159 to 241 days at baseline. After six months, there was a decrease of 100 and 114 waiting days in two localities, respectively, with reduction of waiting times only for high-risk cases in the third locality. CONCLUSIONS: Adoption of telehealth strategies resulted in the reduction of number of cases on waiting lists. Results were consistent across localities, suggesting that telehealth interventions are viable in diverse settings.

Mapeamento dos domínios da ANVISA para o padrão ISO IDMP / Mapping of ANVISA domains to the ISO IDMP standard / Mapeo de dominios de ANVISA al estándar ISO IDMP

Objetivo: Apresentar o mapeamento entre vocabulários controlados da Agência Nacional de Vigilância Sanitária (ANVISA) para listas do European Directorate for the Quality of Medicines and Healthcare (EDQM). Método: O mapeamento obedeceu aos princípios da ABNT NBR ISO 12300. Resultados: Foram mapeadas as listas: Via de Administração, Forma Farmacêutica e Embalagem. 47% dos mapeamentos foram classificados com grau de equivalência 4, onde o conceito fonte foi mais restrito com mais significado específico que o conceito/termo alvo. Conclusão: Entende-se que este estudo fornece subsídios para a ANVISA prosseguir no trabalho de harmonização das listas locais com o padrão IDMP.

Objective: To present the mapping between controlled Brazilian Health Regulatory Agency (ANVISA) vocabularies for European Directorate for the Quality of Medicines and Healthcare (EDQM) lists. Method: The mapping followed the principles described in the ABNT NBR ISO 12300. Results: Terms of three lists were mapped: Routes of Administration, Pharmaceutical Dose Forms and Packaging. Almost half of the mappings were classified with equivalence grade 4 meaning that the source concept was more restricted with more specific meaning than the target concept / term. Conclusion: This work provides the necessary subsidies for ANVISA to proceed with the work of harmonizing local lists with the IDMP standard.

Objetivo: Presentar el mapeo entre vocabularios controlados de Agencia Nacional de Vigilancia Sanitaria (ANVISA) para listas European Directorate for the Quality of Medicines and Healthcare (EDQM). Método: El mapeo siguió los principios descritos en lo estándar ABNT NBR ISO 12300. Resultados: Se mapearon los términos de las listas: Vía de administración, Forma farmacéutica y Embalajes. La mayoría se clasificaron como grado de equivalencia 4, donde el concepto fuente era más restringido con un significado más específico que el concepto/término objetivo. Conclusión: Se entiende que este estudio proporciona subsidios para ANVISA continúe el trabajo de armonizar las listas locales con el estándar IDMP.

Reactivation of latent infections in solid organ transplant recipients from sub-Saharan Africa: What should be remembered?

International migration from Sub-Saharan African countries to the European Union and the United States has significantly increased over the past decades. Although the vast majority of these immigrants are young and healthy people, a minority can be affected by chronic conditions eventually leading to solid organ transplantation (SOT). Importantly, these candidates can bear geographically restricted fungal and parasitic latent infections that can reactivate after the procedure. An appropriate evaluation before transplantation followed by treatment, whenever necessary, is essential to minimize such risk, as covered in the present review. In short, infection due to helminths (Schistosoma spp. and Strongyloides stercoralis) and intestinal protozoa (Entamoeba histolytica, Giardia lamblia or Cyclospora cayetanensis) can be diagnosed by multiple direct stool examination, serological assays and stool antigen testing. Leishmaniasis can be assessed by means of serology, followed by nucleic acid amplification testing (NAAT) if the former test is positive. Submicroscopic malaria should be ruled out by NAAT. Screening for Histoplasma spp. or Cryptococcus spp. is not routinely indicated. Consultation with an Infectious Diseases specialist is recommended in order to adjust preemptive treatment among Sub-Saharan African SOT candidates and recipients.

Expression of Thomsen-Friedenreich Antigen in Colorectal Cancer and Association with Microsatellite Instability.

Microsatellite instability (MSI) is a molecular phenotype due to a deficient DNA mismatch repair (dMMR). In colorectal cancer (CRC), dMMR/MSI is associated with several clinical and histopathological features, influences prognosis, and is a predictive factor of response to therapy. In daily practice, dMMR/MSI profiles are identified by immunohistochemistry and/or multiplex PCR. The Thomsen-Friedenreich (TF) antigen was previously found to be a potential single marker to identify MSI-high gastric cancers. Therefore, in this study, we aimed to disclose a possible association between TF expression and MSI status in CRC. Furthermore, we evaluated the relationship between TF expression and other clinicopathological features, including patient survival. We evaluated the expression of the TF antigen in a cohort of 25 MSI-high and 71 microsatellite stable (MSS) CRCs. No association was observed between the expression of the TF antigen and MSI-high status in CRC. The survival analysis revealed that patients with MSI-high CRC showed improved survival when the TF antigen was expressed. This finding holds promise as it indicates the potential use of the TF antigen as a biomarker of better prognosis in MSI-high CRCs that should be validated in an independent and larger CRC cohort.

Pulmonary mucormycosis: A case report.

Mucormycosis is a rare fungal infection caused by Mucorales order fungi. The rhino-cerebral form of mucormycosis is most commonly seen in patients with diabetes mellitus, whereas, pulmonary mucormycosis is a rare manifestation in patients with haematological malignancy and transplant recipients. We report a case of pulmonary mucormycosis presenting with a late acute onset diabetes on a patient immunosuppressed with a low dose of steroids. We aim to illustrate the need for a high clinical suspicion for the diagnosis of mucormycosis and to report the importance of early and aggressive inhiation of antifungal therapy.

Panton-Valentine Leukocidin-Producing Staphylococcus aureus Infection: A Case Series.

Panton-Valentine leukocidin-producing Staphylococcus aureus (PVL-SA) is associated with relapsing multifocal skin and soft tissue infections (SSTI), necrotizing pneumonia (NP) and severe musculoskeletal infections. Epidemiology is underknown and underdiagnosis is likely. Recent travel abroad, case clustering and relapsing disease are often reported. We reviewed all cases of PVL-SA infection diagnosed at our center, and found 21 cases over a 43-month period. Most patients were adult males, had relevant travel history, reported recurrent disease and presented with SSTI. Etiologic diagnosis took up to five years; meanwhile, 42% of patients had antibiotic treatments. Draining procedures were required in 43% of patients and intensive care support in 19%. All patients recovered. Methicillin-resistance prevalence was 24%. Only 2/13 decolonized patients had posterior relapsing SSTI, both with likely infected contacts. PVL-SA infection's severity and impact are clear, even in small case series as ours. Physician awareness and active PVL-gene search are crucial for an adequate management.

Azole-resistant Candida albicans Spondylodiscitis After Bariatric Surgery: A Case Report.

CASE: We present a case of azole and partial caspofungin-resistant Candida albicans spondylodiscitis, after bariatric surgery with bowel perforation. Treatment included debridement and several months of anidulafungin, complemented with antibacterial therapy because of relapse for bacterial superinfection. After treatment, the infection did not recur clinically or radiologically during one and half years follow-up. CONCLUSION: Although C. albicans spondylodiscitis is rare, fungi should be suspected as a causative agent. Adequate history, imaging and laboratory testing, and medical and surgical treatment should be performed to successfully eradicate the infection and resolve potential neurological deficits.

Prospective study of blood viral load of Epstein-Barr virus, herpes virus type 6 and human parvovirus B19 in Crohn's disease: Does therapy matter?

BACKGROUND: The interplay between inflammatory bowel disease (IBD) and DNA viruses, such as Epstein-Barr (EBV), human parvovirus B19 (HPVB19) and human herpes type 6 (HHV6) is scarcely studied. The main aim of this prospective study is to screen for EBV, HSV6, and HPVB19 DNA viremia in adult patients with stable Crohn's disease (CD), correlating the results with IBD treatment. METHODS: From July 2015 - March 2017, 100 patients were enrolled and divided in four groups of 25 participants each, according to in course treatment. Blood collections were performed every 5 months in all patients. Antibodies for EBV and HPVB19 were screened and repeated if negative. Blood EBV DNA, HPVB19 DNA, and HHV6 DNA were quantified by quantitative real-time Polymerase Chain Reaction. RESULTS: Patients had evidence of EBV (100 %) and HPVB19 (70 %) past infection. Across the study timeline, EBV-DNA, HPVB19-DNA, and HHV6-DNA were detected in the blood of 25, 11, and 7 patients, respectively. Viremia was detected only once in 72 %, 73 %, and 86 % of the patients in the studied period, for EBV, HPVB19, and HHV6, respectively. We did not find significant differences between treatment groups, independently of the viral cut-off for the three viruses. CONCLUSIONS: The detection of EBV, HPVB19, and HHV6 viremia, in stable CD patients, was not impacted by biological/immunosuppressant therapy. Although attractive as a non-invasive technique, this approach did not prove to be useful in stable patients. More and larger studies are needed to address the relevance of these viruses on IBD course, in stable patients and during exacerbations.

Mucormycosis: Literature review and retrospective report of 15 cases from Portugal.

BACKGROUND AND PURPOSE: Prevalence of mucormycosis is growing with the increase of the population at risk. Current recommendations for its management are mostly based on retrospective studies. 3 study aimed to present the cumulative experience of an Infectious Diseases Department from a Portuguese hospital in the management of mucormycosis and discuss the potential gaps in the diagnostic and therapeutic approaches of this infection. MATERIALS AND METHODS: For the purposes of the study, the electronic hospital database was searched for adult patients with mucormycosis from 1996 to 2019 based on the definition provided by the Consensus Definitions of Invasive Fungal Disease. Demographic, clinical, treatment, and outcome data were collected and compared to what had been described in the related literature. RESULTS: In total, 15 cases of mucormycosis were found, including 11 cases with sinus involvement (10 with central nervous system involvement), two pulmonary, and two gastrointestinal infections. Diabetes mellitus (n=7) and corticosteroid therapy (n=7) were frequent risk factors. Median duration of symptoms before the suspicion of diagnosis was 26 days (3-158). The diagnosis was confirmed in 12 patients mostly by histopathology (n=9); the culture was positive only once. Systemic antifungals and surgical debridement were the backbones of treatment; however, side effects, the need for therapeutic drug monitoring, and the anatomical location of lesions added complexity to management. Overall, seven patients died, two of them before the consideration of clinical suspicion. CONCLUSION: More medications are becoming available for the treatment of mucormycosis. Nevertheless, we believe that its prognosis will only significantly change through the increase of awareness and reduction of the time to diagnosis. An effective multidisciplinary approach among surgeons, infectious diseases specialists, radiologists, microbiologists, and anatomopathologists is critical to the achievement of this goal.

Asymptomatic reactivation of hepatitis B virus after prolonged treatment with etanercept.

Etanercept is an anti-tumor necrosis factor ɑ (anti-TNFɑ) drug used for treating immunomediated inflammatory diseases. It is least associated with hepatitis B virus (HBV) reactivation. We present a 71-year-old man with psoriasis refractory to phototherapy and acitretin, which led to etanercept monotherapy. Before anti-TNFɑ treatment, past contact with HBV was elicited; antibodies to HBc and HBs were positive whereas HBsAg was negative. Seven years after treatment initiation, while the patient was completely asymptomatic, a transaminase elevation was found and a reactivation of HBV was documented, with a high viral load of the virus. He started entecavir therapy and, after a 14-month follow-up, the viral load is still detectable at a low level, as well as HBsAg. We emphasize the late and asymptomatic reactivation of HBV associated with soluble anti-TNFɑ monotherapy. This case reinforces the importance of current recommendations for periodic monitoring of viral load and HBV markers in all patients that have had prior contact with HBV (positive anti-HBc), with or without indication for treatment of HBV (HBsAg and HBV-DNA negative).

Comparison between in vitro embryo production using Y-sorted sperm and timed artificial insemination with non-sorted sperm to produce crossbred calves.

Due to the increasing use of in vitro embryo production (IVEP) and the importance of crossbreeding for beef production, pregnancy rates of Nelore recipients were evaluated following Fixed Time Embryo Transfer with fresh or vitrified IVEP embryos produced with Y-sorted sperm of Angus bulls (B. taurus) or Fixed Time Artificial Insemination using non-sorted sperm. For IVEP in Experiment 1, oocytes were obtained using Ovum Pick Up (OPU) (n = 84 embryos) or from ovaries from a slaughterhouse (SLAUGHTER, n = 66 embryos). In Experiment 2, with oocytes obtained by OPU, IVEP embryos were fresh (FRESH, n = 271) or after vitrification/warming (VITRIFIED, n = 79) and PR was compared with FTAI (n = 239). In Experiment 1, cleavage rates were 63.8% and 39.1% for OPU and SLAUGHTER groups, respectively (P = 0.02), and blastocyst rates were 30.5% and 14.7%, respectively (P = 0.09). The PR was similar when considering the source of oocytes (OPU = 35.7%; SLAUGHTER = 25.8%; P = 0.17). In Experiment 2, there was no difference in PR for FRESH or VITRIFIED embryos (34.3% and 30.4%, respectively, P = 0.72), but lesser than FTAI (47.7, P = 0.002). It is concluded that the IVEP with Y-sorted sperm associated with vitrification or embryos produced with oocytes from different sources did not affect PR when there was transfer of crossbred embryos into recipients, and can optimize large-scale application of IVEP technology; however, FTAI pregnancy rates with non-sex sorted sperm were greater.

Chronic meningococcemia.

The spectrum of Neisseria meningitidis-associated clinical entities involves mild forms of disease, without neurological involvement or sepsis, and asymptomatic carrier states. Rarely, N. meningitidis bacteremia can be associated with a prolonged fever with or without arthritis, which we designate as chronic meningococcemia. Chronic meningococcemia is an uncommon entity, usually associated to serogroup B N. meningitidis. Diagnosis is frequently delayed as blood cultures collected outside febrile periods can be negative. We present a case of chronic meningococcemia in a 22-year-old woman with no relevant clinical background, presenting with fever, arthralgia and exanthem. Due to the potential for progression to more severe disease and the risk of N. meningitidis transmission and development of secondary cases, a high degree of clinical suspicion is required to ensure prompt recognition and adequate treatment. Our patient had a favorable outcome probably due to early recognition and adequate treatment, which is critical for the resolution of the disease without complications.

Delaying meiotic resumption during transportation of bovine cumulus-oocyte complexes: effects on development, apoptosis and caspases activity of in vitro-produced embryos.

This study examined the effects of meiosis inhibition during bovine oocyte transportation on developmental competence and quality of produced embryos. The transportation medium was supplemented with: 100 µM butyrolactone I (BL), 500 µM IBMX + 100 µM forskolin (mSPOM), 100 µM milrinone (MR) or follicular fluid (bFF), and was carried out in a portable incubator for 6 h. Next, oocytes were in vitro matured (IVM) for 18 h, without the meiotic inhibitors, with the exception of mSPOM group, in which was added 20 µM cilostamide. The three control groups were IVM with 10% fetal calf serum (FCS) (Control Lab FCS) or 0.6% bovine serum albumin (BSA) (Control Lab BSA) in a CO2 in air incubator or in the portable incubator with 0.6% BSA (Control Transp BSA). Higher cleavage rates (P 0.05) to the other groups (23.6 ± 3.3% to 28.8 ± 2.7%). The total number of blastomeres was higher (P 0.05) from bFF (67.7 ± 4.2). No differences (P > 0.05) were found in apoptosis by the activity of caspases (139.0 ± 3.2 to 152.4 ± 6.5, expressed in fluorescence intensity) as well as the percentage of TUNEL-positive cells (12.3 ± 2.0% to 15.7 ± 1.7%). In conclusion, the transportation of oocytes over 6 h with BL, mSPOM or bFF enabled the acquisition of developmental competence at similar rates to the Control Lab FCS group.

Consequences of endogenous and exogenous WNT signaling for development of the preimplantation bovine embryo.

The specific role of WNT signaling during preimplantation development remains unclear. Here, we evaluated consequences of activation and inhibition of ß-catenin (CTNNB1)-dependent and -independent WNT signaling in the bovine preimplantation embryo. Activation of CTNNB1-mediated WNT signaling by the agonist 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) and a glycogen synthase kinase 3 inhibitor reduced development to the blastocyst stage. Moreover, the antagonist of WNT signaling, dickkopf-related protein 1 (DKK1), alleviated the negative effect of AMBMP on development via reduction of CTNNB1. Based on labeling for phospho c-Jun N-terminal kinase, there was no evidence that DKK1 activated the planar cell polarity (PCP) pathway. Inhibition of secretion of endogenous WNTs did not affect development but increased number of cells in the inner cell mass (ICM). In contrast, DKK1 did not affect number of ICM or trophectoderm (TE) cells, suggesting that embryo-derived WNTs regulate ICM proliferation through a mechanism independent of CTNNB1. In addition, DKK1 did not affect the number of cells positive for the transcription factor yes-associated protein 1 (YAP1) involved in TE formation. In fact, DKK1 decreased YAP1. In contrast, exposure of embryos to WNT family member 7A (WNT7A) improved blastocyst development, inhibited the PCP pathway, and did not affect amounts of CTNNB1. Results indicate that embryo-derived WNTs are dispensable for blastocyst formation but participate in regulation of ICM proliferation, likely through a mechanism independent of CTNNB1. The response to AMBMP and WNT7A leads to the hypothesis that maternally derived WNTs can play a positive or negative role in regulation of preimplantation development.

Lipid content and cryotolerance of in vitro-produced bovine embryos treated with forskolin before vitrification / Acúmulo de lipídios intracitoplasmáticos, desenvolvimento e criotolerância de embriões bovinos produzidos in vitro e tratados com diferentes concentrações de forskolin antes da vitrificação

The aim of the present study was to evaluate the intracytoplasmic lipid content, development and cryotolerance of in vitro-produced bovine embryos treated with different concentrations of forskolin before vitrification. Embryos were produced from abattoir-derived ovaries and allocated into four groups. In the treatment groups, forskolin was added to the in vitro culture medium on Day 6 and incubated for 24 hours in one of the following concentrations: 2.5µM (Forsk 2.5 group), 5.0µM (Forsk 5.0 group) or 10.0µM (Forsk 10.0 group). Embryos from the control group were cultured without forskolin. On Day 7 of culture, the expanded blastocysts were stained with the lipophilic dye Sudan Black B for determination of the intracytoplasmic lipid content or were cryopreserved via the Vitri-Ingá® procedure. Although there were no significant differences (P>0.05) in the blastocyst rates between the Control group (44.9%) and the other treatments, the embryo production was lower (P<0.05) in Forsk 10.0 group (38.8%) compared to Forsk 2.5 (50.5%) and Forsk 5.0 (54.7%) groups. The intracytoplasmic lipid content (expressed in arbitrary units of pixels) in blastocysts from the Control group (1.00±0.03) was similar (P>0.05) to that found in Forsk 2.5 (0.92±0.03) and Forsk 10.0 groups (1.06±0.03) groups; however the lipid accumulation in blastocysts from Forsk 5.0 group (0.82±0.04) was lower than in the Control group (P<0.05). Based on these results, Forsk 5.0 treatment was tested for cryotolerance and it was observed that the blastocoel re-expansion rate evaluated 24 hours after warming was greater (P<0.05) in Forsk 5.0 group (72.2%) compared to the Control group (46.2%). In conclusion, pre-treatment with forskolin at a concentration of 5.0 µM for 24 hours before vitrification is effective in reducing the intracytoplasmic lipid content and, consequently, improves cryotolerance of IVP bovine embryos.(AU)

Os embriões foram produzidos a partir de ovários obtidos em abatedouro e foram alocados em quatro grupos experimentais. Nos grupos tratados, o forskolin foi adicionado ao meio de cultivo in vitro no dia 6 do cultivo e os embriões foram incubados durante 24 horas com uma das seguintes concentrações: 2,5µM (grupo Forsk 2,5), 5,0µM (grupo Forsk 5,0) ou 10,0µM (grupo Forsk 10,0). Os embriões do grupo controle foram cultivados na ausência de forskolin. No dia 7 do cultivo, os blastocistos expandidos foram corados com o corante lipofílico Sudan Black B para a determinação do teor de lípidos intracitoplasmáticos ou foram criopreservados através do protocolo Vitri-Ingá®. Não foi observada diferença significativa (P>0,05) na taxa de produção de blastocistos entre o grupo Controle (44,9%) e os demais tratamentos, todavia observou-se menor produção de embriões (P<0,05) no grupo Forsk 10,0 (38,8%) em comparação com os grupos Forsk 2,5 (50,5%) e Forsk 5,0 (54,7%). A quantidade de lipídeos intracitoplasmáticos do grupo Controle (1,00±0,03) foi semelhante (P>0,05) a dos grupos Forsk 2,5 (0,92±0,03) e Forsk 10,0 (1,06±0,03); no entanto, o acúmulo de lípidos nos blastocistos do grupo Forsk 5.0 (0,82 ± 0,04) foi menor do que no grupo controle (P<0,05). A partir destes resultados, o grupo Forsk 5,0 foi testado quanto à criotolerância e foi observado que a taxa de re-expansão da blastocele 24 horas após o aquecimento foi maior (P<0,05) no grupo Forsk 5,0 (72,2%) quando comparado ao grupo Controle (46,2%). Em conclusão, o pré-tratamento com forskolin na concentração de 5,0 µM durante 24 horas antes da vitrificação foi eficiente para promover a redução da quantidade de lipídeos intracitoplasmáticos e, consequentemente, melhorou a criotolerância de embriões bovinos produzidos in vitro.(AU)

Membrane lipid profile of in vitro-produced embryos is affected by vitrification but not by long-term dietary supplementation of polyunsaturated fatty acids for oocyte donor beef heifers.

Dietary rumen-protected polyunsaturated fatty acids (PUFAs) rich in linoleic acid (LA) may affect embryo yield, and LA can modulate the molecular mechanisms of lipid uptake in bovine blastocysts produced in vitro. In embryos, membrane lipids, such as phosphatidylcholines (PCs) and sphingomyelins (SMs), affect cryopreservation success. The aim of the present study was to evaluate embryonic developmental rates after the IVF of oocytes retrieved from Nellore heifers fed for approximately 90 days with rumen-protected PUFAs rich in LA. In addition, we evaluated embryo cryotolerance and the membrane structure lipid composition using matrix-assisted laser desorption ionisation mass spectrometry of fresh and vitrified embryos. Embryo development to the blastocyst stage (mean 43.2%) and embryo survival after vitrification and warming (mean 79.3%) were unaffected by diet. The relative abundance of one lipid species (PC ether (PCe; 38:2, which means that this lipid has 38 carbon atoms and 2 double bonds in the fatty acyl residues) was increased after PUFAs supplementation. However, 10 ions were affected by cryopreservation; ions consistent with PC 32:0, PC 34:1, SM 24:1, PC 40:6 or PC 42:9, PC plasmalogen (PCp) 44:10 or PC 42:7, triacylglycerol (TAG) 54:9 and a not assigned ion (m/z 833.2) were lower in blastocysts that survived to the cryopreservation process compared with fresh blastocysts, whereas the abundance of the ions PC 36:3 or PC 34:0, PCe 38:2 or PC 36:6 and PC 36:5 or PCe 38:1 were increased after cryopreservation. Thus, the results demonstrate that the mass spectrometry profiles of PC, SM and TAG species differ significantly in bovine blastocysts upon cryopreservation. Because the lipid ion abundances of fresh and vitrified-warmed embryos were distinct, they can be used as potential markers of post-cryopreservation embryonic survival.