Effect of Oil Overlay on Inhibition Potential of Roscovitine in Sheep Cumulus‐Oocyte Complexes

Inhibitors of cyclin‐dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus‐oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 μm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor‐free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (?) at 38.5°C and 5% CO₂. At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/?) exhibited total expansion while in both Rosco groups (+/?) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p < 0.05). This meiotic arrest was reversible, and proper meiosis progression also occurred in the Control groups (+/?). So, the culture system without oil overlay improved the meiotic inhibition promoted by roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression.     

The use of the rapid osmotic fragility test as an additional test to diagnose canine immune-mediated haemolytic anaemia

Background

Diagnosing canine immune-mediated haemolytic anaemia (IMHA) is often challenging because all currently available tests have their limitations. Dogs with IMHA often have an increased erythrocyte osmotic fragility (OF), a characteristic that is sometimes used in the diagnosis of IMHA. Since the classic osmotic fragility test (COFT) is time-consuming and requires specialized equipment, an easy and less labour-intensive rapid osmotic fragility test (ROFT) has been used in some countries, but its diagnostic value has not yet been investigated.This study aimed to evaluate erythrocyte osmotic fragility in dogs with and without IMHA, to compare results of the classic (COFT) and rapid (ROFT) test and to assess the value of the ROFT as diagnostic test for canine IMHA.Nineteen dogs with IMHA (group 1a), 21 anaemic dogs without IMHA (group 1b), 8 dogs with microcytosis (group 2), 13 hyperlipemic dogs (group 3), 10 dogs with lymphoma (group 4), 8 dogs with an infection (group 5) and 13 healthy dogs (group 6) were included.In all dogs, blood smear examination, in-saline auto-agglutination test, Coombs’ test, COFT and ROFT were performed. In the COFT, OF5, OF50 and OF90 were defined as the NaCl concentrations at which respectively 5, 50 and 90% of erythrocytes were haemolysed.

Results

Compared with healthy dogs, OF5 and OF50 were significantly higher in group 1a (P < 0.001) and OF5 was significantly higher in group 3 (P = 0.0266). The ROFT was positive in 17 dogs with IMHA, 10 hyperlipemic dogs, one anaemic dog without IMHA and one healthy dog.

Conclusions

Osmotic fragility was increased in the majority of dogs with IMHA and in dogs with hyperlipidemia, but not in dogs with microcytosis, lymphoma or an infection. Although more detailed information was obtained about the osmotic fragility by using the COFT, the COFT and ROFT gave similar results. The ROFT does not require specialized equipment, is rapid and easy to perform and can be used easily in daily practice. Although, the ROFT cannot replace other diagnostic tests, it may be a valuable additional tool to diagnose canine IMHA.     

Nitric oxide impacts bovine sperm capacitation in a cGMP‐dependent and cGMP‐independent manner

We aimed to elucidate whether NO acts in in vitro sperm capacitation in bovine via cGMP/PKG1 pathway. For this, cryopreserved bovine sperm were capacitated in vitro with 20 µg/ml heparin (Control) plus treatments: 1 mM L‐arginine (L‐arg, NO precursor), 50 µM Rp‐8‐Bromo‐β‐phenyl‐1,N²‐ethenoguanosine‐3′,5′‐cyclic monophosphorothioate (Rp‐8‐Br‐cGMPS, selective inhibitor of the binding site for cGMP in PKG1), 1 mM 2‐Phenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl 3‐oxide (PTIO, NO scavenger), and the combinations of L‐arg + RP‐8‐Br‐cGMPS and L‐arg + PTIO. Sperm motility and vigour were determined by phase‐contrast microscopy, capacitation status by chlortetracycline staining, and the intracellular concentration of cGMP was measured by ELISA. Data were subjected to analysis of variance and means compared with SNK test at 5% probability. Motility and vigour were lower in sperm treated with PTIO when compared to Control and other treatments (p < .05). The L‐arg treatment showed the highest percentage of capacitated sperm when compared to the Control and other treatments (Rp‐8‐Br‐cGMPS, L‐arg + Rp‐8‐Br‐cGMPS and PTIO) (69.8 ± 3.4%, 51.2 ± 3.0, 51.1 ± 2.1, 51.2 ± 3.0 and 45.5 ± 2.7, respectively) (p < .05). The capacitation ratio (%) was lower in treatments with Rp‐8‐Br‐cGMPS, L‐arg + Rp‐8‐Br‐cGMPS and PTIO, respectively (p < .05). Lastly, cGMP concentration (pmol/ml) was lower in PTIO and L‐arg + PTIO (1.3 ± 0.3 and 1.6 ± 0.4) and was higher in Rp‐8‐Br‐cGMPS and L‐arg + Rp‐8‐Br‐cGMPS (3.7 ± 0.4 and 4.0 ± 0.5) treatments. We showed that during in vitro capacitation of cattle: (a) NO influences sperm motility and vigour; (b) NO is associated with cGMP synthesis through two independent pathways and (c) the cGMP/PKG1 pathway has a partial role in sperm capacitation and does not involve the L‐arg/NO.     

Comparison of serological assays for the diagnosis of canine visceral leishmaniasis in animals presenting different clinical manifestations

Three serological methods, indirect fluorescent immunoassay (IFI), enzyme-linked immunosorbent assay (ELISA) and direct agglutination test (DAT) that are commonly employed in the diagnosis of canine visceral leishmaniasis (CVL), have been assessed. A total of 234 domestic dogs, drawn from an area in the municipality of Belo Horizonte, Minas Gerais, Brazil, endemic for visceral leishmaniasis, were submitted to clinical and parasitological examinations and serological assay. Sera collected from confirmed non-infected dogs (n=20), and from dogs with other parasitic diseases including Trypanosoma cruzi (n=7), Leishmania braziliensis (n=5), Toxoplasma gondii (n=5) and Ehrlichia canis (n=3), were also included in the study. IFI presented a lower sensitivity (72%) than ELISA (95%), although the specificities of these assays were low (52 and 64%, respectively) and both exhibited cross-reactivity with sera from dogs infected with T. cruzi, L. braziliensis and E. canis. In contrast, DAT exhibited a high sensitivity (93%) and a high specificity (95%) and cross-reacted with only one serum sample derived from an E. canis-infected dog. The reproducibilities of the ELISA and DAT assays were excellent, whilst that of IFI was considered to be acceptable. The results produced by ELISA and DAT were in complete agreement, those between ELISA and IFI were at an acceptable level of agreement, whilst the concurrence between the IFI and DAT results were either acceptable or poor depending on the clinical conditions of the group of dogs examined. Since there is no readily accessible method for the diagnosis of CVL that offers 100% specificity and sensitivity, the choice of technique employed must depend on the aim of the investigation.     

Sensitivity of Soil P Availability Tests to Ca-P in Oxisols

We assessed the sensitivity of mixed exchange resin (MER) and Mehlich-3 (M3) to calcium-bound phosphate (Ca-P) in Oxisols. These methods were compared with Mehlich-1 (M1) and anion exchange resin (AER), which have high and low sensitivity, respectively, to this soil P form. We used maize (Zea mays L.) as a test plant. Three samples from two Oxisols were treated with five ratios of Triple Superphosphate—TS and Bayóvar Phosphate Rock—BPR (0 + 100; 25 + 75, 50 + 50, 75 + 25 and 100 + 0% TS + BPR). The MER extracted more P than AER and M3 at the highest BPR:TS ratios (high Ca-P) and similar quantities at the higher TS:BPR ratios (low Ca-P) for two soil samples. Unlike M1-extracted P, M3-P, AER-P, and MER-P positively correlated with maize dry matter production and P uptake. Mehlich-3 and MER were suitable to assess P availability in high Ca-P Oxisols.     

Effect of recombinant human TSH on the uptake of radioactive iodine (I) by the thyroid gland in healthy beagles

In human medicine, recombinant human thyroid-stimulating hormone (rhTSH) increases thyroid radioactive iodine uptake (RAIU), allowing radioiodine-131 (¹³¹I) dose reduction and greater efficacy in the treatment of differentiated thyroid cancer and multinodular goiter. The goal of this study was to evaluate the effect of rhTSH, administered 24 h and 48 h before radioiodine-123 (¹²³I), on the thyroid RAIU in healthy dogs. Seven healthy euthyroid beagles were randomly allocated to 3 groups (2 groups of 2 dogs and 1 group of 3 dogs) in a prospective, blinded, crossover study. At Week 1, 1 group received ¹²³I for a baseline RAIU; 1 group received 100 μg of rhTSH IV 24 h before ¹²³I, and 1 group received 100 μg of rhTSH IV 48 h before ¹²³I. All dogs received 37 MBq of radioactive ¹²³I IV, and thyroid RAIU was determined 8 h, 24 h, and 48 h thereafter. The study was designed in such a manner that each dog received the 3 treatments and a wash-out period of 3 wk was respected in between. Blood samples were taken for measurement of serum total thyroxine (TT4) and thyrotropin (TSH) concentrations at baseline and 6 h, 12 h, 24 h, and 48 h after rhTSH administration. Recombinant human TSH caused no significant change on thyroid RAIU. The overall mean thyroid RAIU significantly decreased during the study independent of the treatment. Recombinant human TSH significantly increased serum TT4 concentration, which peaked 6 h after rhTSH administration. Compared to baseline, serum TSH concentration remained higher at 6 h, 12 h, 24 h, and 48 h. However, a statistically significant difference was reached only at 6 h and 12 h after rhTSH administration. No adverse effects of rhTSH were observed during the study. Further studies are needed to determine the best timing and dosage of administration of rhTSH in healthy and thyroid carcinoma dogs.     

Effects of temperature on growth,survival and physiological parameters in juveniles of Lophiosilurus alexandri,a carnivorous neotropical catfish

This study aimed to analyse the effects of different water temperatures on the growth, survival and blood physiological parameters of Lophiosilurus alexandri. Juveniles measuring 12.6 ± 0.5 cm and weighing 30.6 ± 3.6 g were subjected to four water temperature regimes: 23, 26, 29 and 32°C with four replicates. The animals were fed twice daily with formulated diet. The experiment lasted for 35 days. Survival was 100% at all water temperatures, and the best temperature for growth was estimated to be 27.8°C using a quadratic regression model. The estimated temperatures for the best feed conversion and highest feed consumption rates were 26.2 and 28.8°C respectively. The fat visceral‐somatic index was highest at 32°C. The highest haemoglobin and haematocrit values were 29.4 and 32.1°C respectively. For plasma protein and cholesterol, the lowest values estimated were 23.9 and 24.1°C respectively. Glucose and liver nitrogen content increased with rising temperature. Higher values of total ammonia in the water were observed at higher temperatures. No effects of temperature were seen on the amounts of muscle dry matter, nitrogen, energy content and triglycerides or on the hepatosomatic index. Water temperature has an important role in the developmental and blood physiological parameters of juvenile L. alexandri, and the quadratic regression model showed that the appropriate temperature for growth is between 27 and 28°C.     

Bioremediation of Mangroves Impacted by Petroleum

The majority of oil from oceanic oil spills (e.g. the recent accident in the Gulf of Mexico) converges on coastal ecosystems such as mangroves. Microorganisms are directly involved in biogeochemical cycles as key drivers of the degradation of many carbon sources, including petroleum hydrocarbons. When properly understood and managed, microorganisms provide a wide range of ecosystem services, such as bioremediation, and are a promising alternative for the recovery of impacted environments. Previous studies have been conducted with emphasis on developing and selecting strategies for bioremediation of mangroves, mostly in vitro, with few field applications described in the literature. Many factors can affect the success of bioremediation of oil in mangroves, including the presence and activity of the oil-degrading microorganisms in the sediment, availability and concentration of oil and nutrients, salinity, temperature and oil toxicity. More studies are needed to provide efficient bioremediation strategies to be applicable in large areas of mangroves impacted with oil. A major challenge to mangrove bioremediation is defining pollution levels and measuring recuperation of a mangrove. Typically, chemical parameters of pollution levels, such as polycyclic aromatic hydrocarbons (PAHs), are used but are extremely variable in field measurements. Therefore, meaningful mangrove monitoring strategies must be developed. This review will present the state of the art of bioremediation in oil-contaminated mangroves, new data about the use of different mangrove microcosms with and without tide simulation, the main factors that influence the success of bioremediation in mangroves and new prospects for the use of molecular tools to monitor the bioremediation process. We believe that in some environments, such as mangroves, bioremediation may be the most appropriate approach for cleanup. Because of the peculiarities and heterogeneity of these environments, which hinder the use of other physical and chemical analyses, we suggest that measuring plant recuperation should be considered with reduction in polycyclic aromatic hydrocarbons (PAHs). This is a crucial discussion because these key marine environments are threatened with worldwide disappearance. We highlight the need for and suggest new ways to conserve, protect and restore these environments.     

Biochar as a strategy to manage plant diseases caused by pathogens inhabiting the soil: a critical review

Phytoparasitica - The current agricultural scenario faces a range of challenges, with phytosanitary ones being paramount. In most cases, plant diseases are treated with chemicals; however, they...