Manipulated Mouse Embryos as Bioassay System for Water Quality Control

Mouse pronuclear stage embryos with intact slit zona pellucida (manipulated) were cultured in vitro until the hatched blastocyst stage in simplex optimized medium with higher K⁺ concentration (KSOM) prepared with three different water types: tap, deionized reverse osmosis (D‐O) water and Milli‐Q system (M‐Q) water. The culture media were supplemented with or without protein and ethylenediaminetetraacetic acid (EDTA, disodium salt). The rates of hatched blastocysts were significantly affected (p < 0.01) by micromanipulation, protein supplement and water source. The water source has no influence (p > 0.05) on development in EDTA‐supplemented protein‐free culture media, whereas in EDTA‐ and protein‐free culture media, the water quality significantly (p < 0.001) affected the rates of development, with higher rates in media prepared with M‐Q water. The micromanipulated embryos showed higher sensitivity to the water quality (p < 0.01). It worth mentioning that the rates of hatched blastocysts in protein‐free culture media were very low (0–7.5%). Furthermore, the three different water types were analysed by measuring the electrical conductivity, inorganic ions, total organic carbon and endotoxins to evaluate the purity. M‐Q water showed the lowest levels of inorganic ion, total organic carbon and endotoxin concentrations. We concluded that manipulated mouse embryos are good system to evaluate the quality of water used in biological system.     

Hemodynamic effects of incremental doses of acepromazine in isoflurane-anesthetized dogs

ObjectiveTo evaluate the effects of incremental doses of acepromazine on hemodynamics in isoflurane-anesthetized dogs.Study designProspective, experimental study.AnimalsHealthy, adult, mixed-breed dogs (two male and four female) weighing 16.8 ± 5.1 kg (mean ± standard deviation).MethodsDogs were anesthetized with propofol (7 mg kg^–1) intravenously (IV) and isoflurane. Thermodilution and arterial catheters were placed for hemodynamic monitoring and arterial blood sampling for blood gas analysis. Baseline measurements were performed with stable expired concentration of isoflurane (Fe′Iso) at 1.8%. Each dog was then administered four incremental acepromazine injections (10, 15, 25 and 50 μg kg^–1) IV, and measurements were repeated 20 minutes after each acepromazine injection with Fe′Iso decreased to 1.2%. The four acepromazine injections resulted in cumulative doses of 10, 25, 50 and 100 μg kg^–1 (time points ACP₁₀, ACP₂₅, ACP₅₀ and ACP₁₀₀, respectively).ResultsCompared with baseline, cardiac index (CI) increased significantly by 34%, whereas systemic vascular resistance index (SVRI) decreased by 25% at ACP₅₀ and ACP₁₀₀. Arterial oxygen content (CaO₂) was significantly lower than baseline after all acepromazine injections (maximum decreases of 11%) and was lower at ACP₅₀ and ACP₁₀₀ than at ACP₁₀. No significant change was found in heart rate, stroke index, oxygen delivery index and systolic, mean and diastolic blood pressures. Hypotension (mean arterial pressure < 60 mmHg) was observed in one dog at baseline, ACP₁₀, ACP₂₅ and ACP₁₀₀, and in two dogs at ACP₅₀.Conclusions and clinical relevanceCompared with isoflurane alone, anesthesia with acepromazine–isoflurane resulted in increased CI and decreased SVRI and CaO₂ values. These effects were dose-related, being more pronounced at ACP₅₀ and ACP₁₀₀. Under the conditions of this study, acepromazine administration did not change blood pressure.     

Meta-analysis of fungicide efficacy on soybean target spot and cost–benefit assessment

Target spot of soybean has spread in Brazil, the southeastern United States and Argentina in the last decade. A collaborative network of field Uniform Fungicide Trials (UFT) in Brazil was created in 2011 to study the target spot control efficacy of fungicides, including azoxystrobin + benzovindiflupyr (AZ_BF), carbendazim (CZM), fluxapyroxad + pyraclostrobin (FLUX_PYRA), epoxiconazole + FLUX_PYRA (EPO_FLUX_PYRA), mancozeb (MZB) and prothioconazole + trifloxystrobin (PROT_TRIF). Network meta-analysis was used to conduct a quantitative synthesis of UFT data collected from 2012 to 2016 and to evaluate the effects of disease pressure (DP, low ≤ 35% target spot severity in the nontreated control < high) and year of experiment on the overall mean efficacy and yield response to each of the tested fungicides. Based on mean percentage control of target spot severity, the tested fungicides fall into three efficacy groups (EG): high EG, FLUX_PYRA (76.2% control relative to the nontreated control) and EPO_FLUX_PYRA (75.7% control); intermediate EG, PROT_TRIF (66.5% control) and low EG, MZB (49.6% control), AZ_BF (46.7% control) and CZM (32.4% control). DP had a significant effect on yield response. At DP_Low, the highest response was due to PROT_TRIF (+342 kg ha⁻¹, +12.8%) and EPO_FLUX_PYRA (+295.5 kg ha⁻¹, +11.2%), whereas at DP_High, EPO_FLUX_PYRA and FLUX_PYRA outperformed the other treatments, with yield responses of 503 kg ha⁻¹ (+20.2%) and 469 kg ha⁻¹ (+19.1%), respectively. The probability of a positive return on fungicide investment ranged from 0.26 to 0.56 at DP_Low and from 0.34 to 0.66 at DP_High.     

Immunohistochemical Expression of Growth Factors in the Follicular Wall of Normal and Cystic Ovaries of Sows

The expression of growth factors was evaluated immunohistochemically in normal and cystic ovaries of sows. The immunohistochemically stained area (IHCSA) was quantified by image analysis to analyse the expression of these proteins in the follicular wall of secondary, tertiary and cystic follicles. IGF‐I immunoreactivity was strong in the granulosa cell layer (GC), moderate in the theca interna (TI) and mild in the theca externa (TE) of the normal follicles. There was severe reduction of the labelling to IGF‐I in the GC of the follicular and luteinized cysts. In the normal follicles, the reactivity for IGF‐II was very similar to pattern noted in IGF‐I. There was reduction of the IHCSAs in the GC of the follicular and luteinized cysts, but the decrease was not significant. The staining of the IGF‐II in the TI and TE of the cysts was increased, in comparison with normal follicles. The IHCSAs for VEGF were higher in the GC and TE of the normal follicles in contrast to TI, but this difference was noted only in the tertiary follicle. The VEGF reactivity increased in the GC of the cysts, in relation to normal follicles. The results of the current study show that the formation of ovarian cysts in sows is associated with alterations in the immunohistochemical expression of some growth factors.     

Methods of inoculation and evaluation of Erwinia psidii in eucalypt

The dieback and wilting caused by Erwinia psidii are emerging eucalypt diseases that have been observed since 2014 in the south and central‐south regions of Brazil. Field observations have shown variability in disease severity resistance among Eucalyptus spp. clones and species. It is hypothesized that this variability is due to genetic resistance. To confirm this hypothesis, inoculations in genetically distinct eucalypt plants are necessary. However, lack of an inoculation method and disease assessment makes difficult to select resistant genotypes for use in commercial plantations or genetic breeding programmes. Three inoculation methods were tested on eight clones of Eucalyptus spp. Among them, inoculum deposition with bacteria‐impregnated toothpick on the axillary buds was the simplest and most effective, capable to reproduce the disease symptoms observed under conditions of natural infection. We also developed a rating scale for disease assessment. Among eight clones tested, only Clone 1 (Eucalyptus saligna) and Clone 2 (Eucalyptus urophylla) were resistant.     

Pronuclear Embryo Yield in Canindé and Saanen Goats for DNA Microinjection

The objective of this study was to examine the effect of donor breed on pronuclear‐stage embryo yield to be used for DNA microinjection in a transgenesis goat program. Twelve Canindé and twelve Saanen goats were heat synchronized using a progestagen‐cloprostenol treatment. Forty‐eight hours before the sponge removal, superovulation was induced with a total administration of 4.4 mg/kg bodyweight NIH‐FSH‐P1, given twice daily in decreasing doses over 3 days. In addition, goats received 100 μg of GnRH and they were hand‐mated at 36 and 48 h after progestagen removal. Embryo recovery was performed by oviduct flushing at 72 h after sponge removal. Embryos were microinjected with a DNA construct and noticeable swelling of the nuclei was the criterion for successful microinjection. The total diameter, cytoplasm diameter, zona pellucida thickness and pronuclei diameter were measured for each microinjected embryo. A higher (p < 0.05) percentage of fertilized ova was observed in Canindé (89.9%) than Saanen (36.2%) goats. In addition, Canindé donors produced a higher percentage of pronuclear embryos when compared with Saanen: 72.5% vs 20.6% (p < 0.05), respectively. Successful microinjection was verified in 96.7% and 73.3% of times in Canindé and Saanen embryos, respectively (p < 0.05). Significant differences were observed for all morphometric parameters except for cytoplasm diameter. In conclusion, under our study experimental conditions, Canindé were more efficient than Saanen goats concerning the pronuclear embryo yield and manipulation. The use of Canindé goats in transgenesis programs could be increase the interest in their breeding and could be contribute to saving them from extinction.