Extracellular vesicles derived from bovine adipose-derived mesenchymal stromal cells enhance in vitro embryo production from lesioned ovaries.

BACKGROUND AND AIMS: Ovum pick-up (OPU) is an intrinsic step of in vitro fertilization procedures. Nevertheless, it can cause ovarian lesions and compromise female fertility in bovines. Recently, we have shown that intraovarian injection of adipose-derived mesenchymal stromal cells (AD-MSCs) effectively preserves ovarian function in bovines. Given that MSC-derived extracellular vesicles (MSC-EVs) have been shown to recapitulate several therapeutic effects attributed to AD-MSCs and that they present logistic and regulatory advantages compared to AD-MSCs, we tested whether MSC-EVs would also be useful to treat OPU-induced lesions. METHODS: MSC-EVs were isolated from the secretome of bovine AD-MSCs, using ultrafiltration (UF) and ultracentrifugation methods. The MSC-EVs were characterized according to concentration and mean particle size, morphology, protein concentration and EV markers, miRNA, mRNA, long noncoding RNA profile, total RNA yield and potential for induction of the proliferation and migration of bovine ovarian stromal cells. We then investigated whether intraovarian injection of MSC-EVs obtained by UF would reduce the negative effects of acute OPU-induced ovarian lesions in bovines. To do so, 20 animals were divided into 4 experimental groups (n = 5), submitted to 4 OPU cycles and different experimental treatments including vehicle only (G1), MSC-EVs produced by 7.5 × 106 AD-MSCs (G2), MSC-EVs produced by 2.5 × 106 AD-MSCs (G3) or 3 doses of MSC-EVs produced by 2.5 × 106 AD-MSCs, injected after OPU sessions 1, 2 and 3 (G4). RESULTS: Characterization of the MSC-EVs revealed that the size of the particles was similar in the different isolation methods; however, the UF method generated a greater MSC-EV yield. MSC-EVs processed by both methods demonstrated a similar ability to promote cell migration and proliferation in ovarian stromal cells. Considering the higher yield and lower complexity of the UF method, UF-MSC-EVs were used in the in vivo experiment. We evaluated three therapeutic regimens for cows subjected to OPU, noting that the group treated with three MSC-EV injections (G4) maintained oocyte production and increased in vitro embryo production, compared to G1, which presented compromised embryo production following the OPU-induced lesions. CONCLUSIONS: MSC-EVs have beneficial effects both on the migration and proliferation of ovarian stromal cells and on the fertility of bovines with follicular puncture injury in vivo.

In-Vitro Growth Kinetics of Mesenchymal Stem Cells in Cytotoxicity Tests Using Low-Diluted Viscum Album.

INTRODUCTION: The use of mesenchymal stem cells (MSC) in cytotoxicity tests is an in-vitro alternative model for predicting initial doses. Homeopathic medicines may stimulate the immune system to combat a pathology effectively and have been used for over two centuries. Viscum album (VA) extracts are widely used in the treatment of cancer, due to their immunomodulatory, cytotoxic and pro-apoptotic properties. OBJECTIVE: This study aimed to evaluate the in-vitro growth kinetics of canine MSC in relation to cytotoxicity, cell differentiation and expression of pluripotentiality markers, using a VA preparation at the D1D2 (1×10-1, 1×10-2 potency (VAD1D2). METHODS: MSC were obtained from adipose tissue sampled from a healthy dog that was undergoing an elective veterinary procedure and with its owner's permission. The experiments were performed in three groups: MSC treated with VAD1D2 or diluent or untreated (control). The cytotoxicity was evaluated by MTT assay. The differentiation was induced in three lineages, and apoptotic cell labeling was performed by an Annexin-V test. RESULTS: At the concentration of 10 µL/mL of VA, the number of cells after in-vitro culture was maintained when compared with the control (untreated) group. A significant and gradual decrease in cell viability was recorded as VA concentrations increased. The apoptosis analysis showed that VA at 20 µL/mL presented absolute percentages of initial apoptosis twice as high as at 10 µL/mL, which was similar to the control (untreated group). CONCLUSION: The results suggest that the use of efficient methods to assess the in-vitro cytotoxicity of VA-based homeopathic medicines using MSC lineages may predict the potential action at different concentrations. These findings demonstrated that VAD1D2 interferes with canine MSC growth kinetics.

Molecular characterization and biofilm-formation analysis of Listeria monocytogenes, Salmonella spp., and Escherichia coli isolated from Brazilian swine slaughterhouses.

This study aimed to verify the presence of Listeria monocytogenes, Salmonella spp., and Escherichia coli in two Brazilian swine slaughterhouses, as well as to perform antibiograms, detect virulence and antimicrobial resistance genes, and evaluate the in vitro biofilm-forming capability of bacterial isolates from these environments. One Salmonella Typhi isolate and 21 E. coli isolates were detected, while L. monocytogenes was not detected. S. Typhi was isolated from the carcass cooling chamber's floor, resistant to several antimicrobials, including nalidixic acid, cefazolin, chloramphenicol, doxycycline, streptomycin, gentamicin, tetracycline, and sulfonamide, and contained resistance genes, such as tet(B), tet(C), tet(M), and ampC. It also showed moderate biofilm-forming capacity at 37°C after incubating for 72 h. The prevalence of the 21 E. coli isolates was also the highest on the carcass cooling chamber floor (three of the four samplings [75%]). The E. coli isolates were resistant to 12 of the 13 tested antimicrobials, and none showed sensitivity to chloramphenicol, an antimicrobial prohibited in animal feed since 2003 in Brazil. The resistance genes MCR-1, MCR-3, sul1, ampC, clmA, cat1, tet(A), tet(B), and blaSHV, as well as the virulence genes stx-1, hlyA, eae, tir α, tir ß, tir γ, and saa were detected in the E. coli isolates. Moreover, 5 (23.8%) and 15 (71.4%) E. coli isolates presented strong and moderate biofilm-forming capacity, respectively. In general, the biofilm-forming capacity increased after incubating for 72 h at 10°C. The biofilm-forming capacity was the lowest after incubating for 24 h at 37°C. Due to the presence of resistance and virulence genes, multi-antimicrobial resistance, and biofilm-forming capacity, the results of this study suggest a risk to the public health as these pathogens are associated with foodborne diseases, which emphasizes the hazard of resistance gene propagation in the environment.

Molecular characterization of Salmonella spp. and Listeria monocytogenes strains from biofilms in cattle and poultry slaughterhouses located in the federal District and State of Goiás, Brazil.

Listeria monocytogenes and Salmonella spp. are considered important foodborne pathogens that are commonly associated with foods of animal origin. The aim of this study was to perform molecular characterization of L. monocytogenes and Salmonella spp. isolated from biofilms of cattle and poultry slaughterhouses located in the Federal District and State of Goiás, Brazil. Fourteen L. monocytogenes isolates and one Salmonella sp. were detected in poultry slaughterhouses. No isolates were detected in cattle slaughterhouses. All L. monocytogenes isolates belonged to lineage II, and 11 different pulsotypes were detected. Pulsed-field gel electrophoresis analysis revealed the dissemination of two strains within one plant, in addition to the regional dissemination of one of them. The Salmonella isolate was identified via whole genome sequencing as Salmonella enterica serovar Minnesota ST548. In the sequence analysis, no premature stop codons were detected in the inlA gene of Listeria. All isolates demonstrated the ability to adhere to Caco-2 cells, while 50% were capable of invading them. Antimicrobial resistance was detected in 57.1% of the L. monocytogenes isolates, and resistance to sulfonamide was the most common feature. The tetC, ermB, and tetM genes were detected, and four isolates were classified as multidrug-resistant. Salmonella sp. was resistant to nine antimicrobials and was classified as multidrug-resistant. Resistance genes qnrB19, blaCMY-2, aac(6')-Iaa, sul2, and tetA, and a mutation in the parC gene were detected. The majority (78.5%) of the L. monocytogenes isolates were capable of forming biofilms after incubation at 37°C for 24 h, and 64.3% were capable of forming biofilms after incubation at 12°C for 168 h. There was no statistical difference in the biofilm-forming capacity under the different evaluated conditions. Salmonella sp. was capable of forming biofilms at both tested temperatures. Biofilm characterization was confirmed by collecting the samples consistently, at the same sampling points, and by assessing biofilm formation in vitro. These results highlight the potential risk of cross-contamination in poultry slaughterhouses and the importance of surveillance and pathogen control maintenance programs within the meat production industry.

Integration of georeferenced and genetic data for the management of biodiversity in sheep genetic resources in Brazil.

There are few animal germplasm/gene bank collections in Brazil, and basic studies are needed to attend the future internal and external demands from international partners. The aim of this work was to validate a "proof of concept" that integrates spatial (georeferenced data) and genetic data regarding the local of origin from 3518 DNA samples from 17 different genetic groups or breeds of sheep in the Brazilian Germplasm bank. Spatialisation shows that not all genetic groups have samples in the bank, and collection is concentrated in the conservation nuclei spread nationwide. Only 21% of states with a specific breed have samples in the gene bank. The mean number of animals sampled per collection was 32, while the mean distance travelled to collect samples was 262 km from the conservation nuclei. For example, the Brazilian Somali were only collected in the conservation nucleus in Ceará State. No samples were collected to date for the Cariri breed, which is recognised by the Brazilian Ministry of Agriculture. Only two farms and one breed in the bank are from the northern region. Of the 27 states, there are samples in the gene bank of sheep from 13, so several states have no samples, requiring collection from herds outside the official system of conservation to make sure that studies using this germplasm realised are not biased. Significant genetic differences are seen above 332 km, which should guide future sampling efforts. Suggestions are given for improving the quantity, quality and diversity of samples in the gene bank.

Clinical and biochemical parameters of COVID-19 patients with prior or active dengue fever.

Originated in Wuhan, China, the coronavirus 19 disease (COVID-19) has quickly spread worldwide, reaching countries that already faced other endemics and epidemics. In Brazil, such a concerning situation includes arboviruses, among which the dengue virus stands out. Here, we determined the rate of SARS-CoV-2/dengue virus co-infection in a total of 178 patients with COVID-19 symtoms admitted into a large public hospital of the Federal District of Brazil. Furthermore, we evaluated whether prior or active dengue virus infection influenced hematological, biochemical, and clinical parameters of such patients. One hundred and twelve (63%) individuals tested positive for COVID-19, of which 43 (38.4%) were co-infected with dengue virus, and 50 (44.6%) had antibodies indicative of previous dengue infection. Co-infected patients showed lower numbers of circulating lymphocytes and monocytes, higher glucose rates, and a worse pulmonary condition. Of note, prior infections with dengue virus did not influence clinical parameters, but active dengue fever resulted in higher hospitalization rate. In conclusion, amid the current complex epidemiological scenario in Brazil, our data support the notion that SARS-CoV-2 and dengue co-infection affects an important percentage of COVID-19 patients and leads to worse clinical parameters, requiring greater attention from health authorities.

Efeito da luz ultravioleta na eclodibilidade e viabilidade de pintos provenientes de ovos férteis sujos / Effect of ultraviolet light in hatchability and viability of chicks from dirty fertile eggs

Considerando a necessidade de se desenvolver estratégias para a incubação de ovos sujos e a demanda de se utilizar métodos renováveis de sanitização, a utilização da luz ultravioleta para desinfecção de ovos incubáveis sujos foi o objetivo desta pesquisa. Foram avaliados ovos sem desinfecção (C-), desinfetados com paraformaldeído (C+), e expostos a tempos diferentes de exposição ultravioleta: UV 3’30”, UV 4’30” e UV 5’30”. Os ovos C(+) e os expostos à UV por 3’30” e 5’30” tiveram uma redução significativa na quantidade de colônias na casca dos ovos em relação ao C(-), variando de 0.64 a 1 logUFC/g. Os tratamentos C(+), UV 3’30” e UV 5’30” foram aplicados para avaliação da incubabilidade dos ovos. A eclodibilidade do tratamento UV 3’30” (70,51%) foi superior ao UV 5’30” (51,16%) e similar ao C(+) (55,81%). Conclui-se que o tratamento UV 3’30” é eficaz na redução da contaminação microbiológica de ovos sujos e não afeta negativamente a eclodibilidade e a viabilidade dos pintinhos nascidos.

Considering the need to develop strategies for dirty eggs incubation and the demand to use renewable methods of sanitation, the use of ultraviolet light for disinfecting hatching dirty eggs was the objective of this research. Eggs not disinfected (C-), disinfected with paraformaldehyde (C+) and exposed to different times of UV radiation (UV 3’30”, UV 4’30” and UV 5’30”) were tested. The eggs C(+) and those exposed to UV for 3’30” and 5’30” presented a significant reduction on the number of colonies of eggshells comparing to C(-), with a 0.64 to 1 logUFC/g variation. The treatments C(+), UV 3’30” and UV 5’30” were applied in an incubation phase. The hatchability of treatment UV 3’30” (70,51%) was superior to UV 5’30” (51,16%) and alike C(+) (55,81%). In conclusion, the treatment UV 3’30” is effective in reducing microbiologic contamination of dirty eggs and do not affect negatively hatchability and quality of chicks.

Hemato-biochemical profile of meat cattle submitted to different types of pre-loading handling and transport times.

Pre-loading handling and conditions of transport are related to welfare, disease risk and product quality of production animals. These steps continue to be one of the major animal management problems in Brazil. This study evaluated the effects of different types of pre-loading handling and road transport times on the haematological and biochemical traits of cattle. Eighteen male cattle were submitted to three travel times (24, 48 and 72 h) in a truck soon after load using different types of pre-loading handling: traditional (rough handling), training (gentle handling) and use of flags to movement cattle. Haematological traits, blood biochemical measures as well as blood and faecal cortisol were analysed in order to assess animal welfare and physiological status. The traditional management showed to be more stressful, also had animals with a greater number of neutrophils and lower numbers of lymphocytes than handling with flags, showing that animals submitted to more stressful situations can have compromised immune system. Serum aspartate aminotransferase concentrations were within the reference levels and when taken together with increased creatine kinase patterns observed indicate muscle damage in traditional management. Decrease in glucose concentrations over time from traditional management to flag management was observed, while fructosamine was increased in traditional management with 72 h of travel. When taken together, all reported factors, immune, enzymatic, energetic and hormonal, indicate that the quality of pre-loading handling and time of transport were determinant for animal welfare, its homeostatic balance and sanitary conditions.

Effects of Dietary Exposure to Sulfamethazine on the Hematological Parameters and Hepatic Oxidative Stress Biomarkers in Nile Tilapia (Oreochromis niloticus).

Sulfamethazine (SMZ) is one of the most commonly used sulfonamide compounds in fish farming, and its physiological effects on fish are unknown. SMZ was administered to juvenile Nile tilapia (Oreochromis niloticus) at a dose level of 422 mg kg(-1) body weight, for a period of 11 days, via medicated feed. Fish were divided into two groups, the control group (CG) and the group fed with SMZ in feed. The administration of SMZ did not alter the erythrograms and leukograms of the Nile tilapia. The SMZ-fed group showed the same hepatic lipid peroxidation (LPO) concentration as the CG. Nonetheless, the oral administration of SMZ raised the hepatic catalase (CAT) and glutathione S-transferase (GST) activities, the increase probably being sufficient to prevent hepatic LPO production. The oral administration of SMZ affects the hepatic GST and CAT activities of Nile tilapia.

Tissue accumulation and urinary excretion of Cr in chromium picolinate (CrPic)-supplemented lambs.

Chromium (Cr) concentrations in liver, kidney, spleen, heart, lymph node, skeletal muscle, bone, testis and urine of lambs were measured to trace the biodistribution and bioaccumulation of Cr after oral supplementation with chromium picolinate (CrPic). Twenty-four Santa Inês lambs were treated with four different concentrations of CrPic: placebo, 0.250, 0.375 and 0.500 mg of CrPic/animal/day for 84 days. The basal diet consisted of Panicum maximum cv Massai hay and concentrate. Cr concentrations were measured by ICP-MS measuring (52)Cr as collected mass. There was a positive linear relationship between dose administered and the accumulation of Cr in the heart, lungs and testis. Urinary excretion of Cr occurred in a time and dose-dependent manner, so the longer or more dietary Cr provided, the greater excretion of the element. As some non-carcass components (such as lungs or heart) are added to bone and visceral meal to feed animals, there is a risk of bioaccumulation and biomagnification due to Cr offered as CrPic in the diet.

Blood Parameters and Toxicity of Chromium Picolinate Oral Supplementation in Lambs.

The effects of oral supplementation of chromium picolinate (CrPic) on various blood parameters and their possible toxicity on the liver, kidneys, lungs, heart, and testis were investigated. Twenty-four Santa Inês (SI) lambs were treated with four different concentrations of CrPic (six animals/treatment): placebo, 0.250, 0.375, and 0.500 mg CrPic/animal/day for 84 days. The basal diet consisted of hay Panicum maximum cv Massai and concentrate. Blood and serum were collected fortnightly for analysis. On day 84, the animals were euthanized, and histopathological analysis in the liver, kidney, heart, lung, and testis was made. The liver and kidney were also submitted to electronic microscopy analysis. Differences between treatments (P < 0.05) were observed for packed cell volume (day 84), hemoglobin (day 84), total plasm protein (day 56 and day 84), and triglycerides (day 70). There was no statistically significant relationship between Cr supplementation and histopathology findings, although some animals treated with supplementary Cr showed morphological changes in the liver, kidney, and testis. Thus, the effectiveness of supplementation with Cr remains in doubt as to its physiological action and toxicity in sheep.

Production traits in F1 and F2 crosses with naturalized hair breed Santa Inês ewes.

The once bred ewe slaughter method proposes the use of female lamb to produce a lamb and then both are slaughtered, increasing income and high quality meat production. Thus, this study evaluated the growth and reproduction performance of ewe lamb from Santa Inês (SI), a naturalized genetic resource, and their crosses (Dorper x Santa Inês (DOR), Texel x Santa Inês (TEX), Ile de France x Santa Inês (ILE)), as well as the survivability and development of their offspring. The animals were weighed monthly from birth to 12-months age. Samples of milk were collected on approximately 30 days of lactation. The physical-chemical analysis of milk was performed. SI females (2.94 kg) had significantly lower birth weight than DOR (3.80 kg) and TEX (3.87 kg). ILE females had higher weaning weight and weight at 12 months than SI females, which reflected in higher daily weight gain (ADG) (108.46 g/day) than TEX and SI. The pregnancy rates at 12 months were ILE (57.14%), TEX (25%), DOR (50%), and SI (28.57%), with TEX and SI differing of ILE and DOR (p = 0.03). Therefore, in semi-confinement and in a once-bred ewe production system using crossbreeding and allying meat production and reproduction, we recommend the use of Dorper and Ile de France breeds for crossbreeding with Santa Inês females. These results demonstrated the useful of a local genetic resource in productive system aiming a low cost meat production.

Effect of alternate and simultaneous grazing on endoparasite infection in sheep and cattle / Efeitos do pastejo alternado e simultâneo sobre a infecção de endoparasitas em ovinos e bovinos

This experiment was carried out on 8 ha of Panicum maximum cv. Tanzania pastures, with rotational grazing consisting of 7 days of occupation and 21 days of rest. Four treatments were evaluated: cattle grazing alone (BOV), sheep grazing alone (OVI), cattle and sheep grazing simultaneously (SIM) and cattle grazing followed by sheep (alternate - ALT). Twenty heifers and 30 male Santa Inês lambs were used. Fecal egg count (FEC) and fecal cultures were carried out. Blood was also collected to examine red and white cell series, total plasma protein (TPP), albumin and hemoglobin. FEC and estimated nematode pathogenicity index in sheep were lower in the SIM treatment. The Haemonchus spp. proportion was higher in isolated grazing systems. For sheep, mixed grazing was shown to reduce endoparasite infection, and SIM was better than ALT. For cattle, no difference between grazing systems was seen. Therefore, simultaneous grazing (sheep and cattle) may be a tool for reducing the need for anthelmintic treatments in sheep.

O experimento foi realizado em 8ha de pasto de Panicum maximum cv. Tanzania, com pastejo rotacionado de 7 dias de ocupação e 21 dias de descanso. Quatro tratamentos foram avaliados: bovinos pastejando isoladamente (BOV), ovinos pastejando isoladamente (OVI), bovinos e ovinos pastejando simultaneamente (SIM), e bovinos pastejando previamente aos ovinos (alternado - ALT). Vinte novilhas e 30 cordeiros Santa Inês foram utilizados. Contagem de ovos nas fezes (FEC) e coproculturas foram realizados. Sangue também foi colhido para examinar a série vermelha e branca, proteínas plasmáticas totais (TPP), albumina e hemoglobina. FEC e índice de patogenicidade estimada de nematoides nos ovinos foram menores no tratamento SIM. A proporção de Haemonchus spp. foi maior nos sistemas isolados de pastejo. Para os ovinos, os sistemas consorciados apresentaram redução na infecção endoparasitária, sendo SIM melhor que ALT. Para os bovinos, nenhuma diferença entre os sistemas de pastejo foi verificado. Entretanto, o pastejo simultâneo (ovinos e bovinos) pode ser uma ferramenta para reduzir a necessidade de tratamentos anti-helmínticos em ovinos.

Multivariate analysis of heat tolerance characteristics in Santa Inês and crossbred lambs in the Federal District of Brazil.

Heat tolerance is an important trait for deciding on which genetic resource to use on farms in tropical regions. Three genetic groups of lambs were evaluated by physical and physiological parameters related to adaptation and multivariate analyses used to test if these traits were efficient in separating the genetic groups and which traits were more important in this separation. Crossbred lambs were more similar to each other than to Santa Inês, in general; but individually there was an overlap between Ile de France and Santa Inês lambs. Size traits had higher canonical correlation with physiological variables, while rectal temperature, heart rate, and hemoglobin were more highly correlated with physical traits. Error rates using only physiological traits were high, suggesting that these are inefficient in separating groups of lambs for heat tolerance, while separation with physical traits was more efficient.

Thermographic evaluation of climatic conditions on lambs from different genetic groups.

In production systems the characterization of genetic resources in relation to their capacity to respond to environmental conditions is necessary. The objective of this study was to evaluate the use of infrared thermography for separation of animals from different genetic groups and determine which phenotypic traits are important for climatic adaptation. A total of 126 suckling lambs from four different genetic groups (Santa Inês--SI, Bergamasca--B, Bergamasca X Santa Inês--BS, and Ile de France X Santa Inês--IL) were used. The animals were divided into two groups, one housed and another in an outside paddock. Thermograph photographs were taken at four-hour intervals over three full days. Temperatures of the nose, skull, neck, fore and rear flanks and rump were measured, as well as coat depth, the density and length of hairs, reflectance and color. The daily temperature range during the experimental period was more than 20°C, with animals experiencing heat (12 h to 15 h) and cold (24 h to 4 h) stress. The three main phenotypic traits that influenced genetic group separation were hair density, height of coat, and length of hairs. Thermograph temperatures were able to detect different responses of the genetic groups to the environment. Therefore, infrared thermography is a promising technique to evaluate the response of animals to the environment and to differentiate between genetic groups.

Effect of alternate and simultaneous grazing on endoparasite infection in sheep and cattle.

This experiment was carried out on 8 ha of Panicum maximum cv. Tanzania pastures, with rotational grazing consisting of 7 days of occupation and 21 days of rest. Four treatments were evaluated: cattle grazing alone (BOV), sheep grazing alone (OVI), cattle and sheep grazing simultaneously (SIM) and cattle grazing followed by sheep (alternate - ALT). Twenty heifers and 30 male Santa Inês lambs were used. Fecal egg count (FEC) and fecal cultures were carried out. Blood was also collected to examine red and white cell series, total plasma protein (TPP), albumin and hemoglobin. FEC and estimated nematode pathogenicity index in sheep were lower in the SIM treatment. The Haemonchus spp. proportion was higher in isolated grazing systems. For sheep, mixed grazing was shown to reduce endoparasite infection, and SIM was better than ALT. For cattle, no difference between grazing systems was seen. Therefore, simultaneous grazing (sheep and cattle) may be a tool for reducing the need for anthelmintic treatments in sheep.