Metabolic signature of spent culture media shows lipid metabolism as a determinant of pregnancy outcomes

In the present study, we investigated the spent culture media of in vitro produced (IVP) bovine embryos which did (group Pregnant) or did not (group Non-pregnant) establish pregnancy after transfer. For that purpose, IVP embryos on D5 were transferred to individual droplets for the last 48 h of culture. Embryos at the blastocyst stage were then transferred to synchronized recipients, while respective culture media drops were collected and evaluated individually. The list of metabolites present in spent culture media was obtained by electrospray ionization mass spectrometry (ESI-MS) and analysed with Metaboanalyst® to characterize the metabolic profile of each group. The spectrometric analysis showed that pathways related to lipid metabolism, particularly fatty acids degradation via beta-oxidation, were more present in the Pregnant group whereas no significant pathway was identified in the group Non-pregnant. By using this method, we were able to identify a metabolic signature in culture media that allows for a better comprehension of preferential metabolic routes taken by the most viable embryos. These findings offer great insights into the biochemistry of embryo development and reveal a potential target for the development of better-quality IVP systems, as well as tools to identify bovine embryos with greater chances to establish and maintain pregnancy.     

Midgut cysteine-proteinase activity in the velvetbean caterpillar (<Emphasis Type="Italic">Anticarsia gemmatalis</Emphasis> (Hübner))

Proteinase inhibitors are currently targeted as potential insect control agents, but adaptation to proteinase inhibitors is a recognized limitation to such approach requiring the understanding of how phytophagous species can cope with such compounds. The velvetbean caterpillar (Anticarsia gemmatalis) is a key pest of soybean and well-adapted to its host proteinase inhibitors, which is rich in serine-proteinase inhibitors, particularly trypsin-like proteinase inhibitors. As the expression of cysteine proteinases in the midgut of the velvetbean caterpillar is a potential adaptation to circumvent its host defense, we assessed and characterized the digestive cysteine-proteinase activity from velvetbean caterpillars. Significant soluble and membrane-bound proteolytic activity was obtained and was consistent with those of cysteine proteinases based on the substrate and inhibitors used for their characterization. The K _m and V _max values obtained were 2.35 ± 0.50 mM and 40.89 ± 6.68 nmol min⁻¹ mg⁻¹ for the soluble proteinases and 0.33 ± 0.03 mM and 24.54 ± 0.67 nmol min⁻¹ mg⁻¹ for the membrane-bound proteinases, range of values also consistent with cysteine proteinases. Therefore, the proteolytic activity observed in the velvetbean caterpillar midgut is consistent with that of cysteine proteinases providing preliminary support for the contention of their potential involvement mitigating the negative effects of serine-protease inhibitors in this species.     

Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs

Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn Leishmania chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The present study attempted to standardize a conventional polymerase chain reaction (cPCR) protocol for the detection of L. infantum DNA in canine spleen samples. Quantitative PCR (qPCR) technique was used to confirm the presence of Leishmania DNA in the canine spleen fragments. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. cPCR protocols for spleen samples were standardized using primers that amplify a 145 bp fragment, located at the parasite kinetoplast minicircle. The genus specificity of the cPCR protocol was assessed by its inability to amplify the DNA of other common canine pathogens, such as Ehrlichia canis, Babesia canis, Toxoplasma gondii and Trypanosoma cruzi. cPCR protocol sensitivity was tested by assessing the reaction detection limit, determined to be 10 fg of L. infantum reference strain DNA, which corresponds to a range of 0.03-0.1 parasites per fragment. Standardized cPCR protocol was used to detect the presence of Leishmania in 45 dog spleen samples. Our results showed that 40% of the spleen fragment cultures were positive for Leishmania parasites, 58% of the dog serum samples tested positive using ELISA, and parasite DNA was detected in 44% using qPCR, while 47% of the spleen samples using cPCR. Diagnostic methods performance was assessed and revealed a better degree of ascertainment for cPCR when compared to other diagnostic methods. The sensitivity of ELISA was 83.3%, qPCR was 83.3%, and cPCR was 88.9%; PPV for ELISA was 57.7%, qPCR was 75% and cPCR was 76.2%; the Kappa coefficients were found to be 0.40 (fair) for ELISA, 0.64 (substantial) for qPCR and 0.68 (substantial) for cPCR. In both oligosymptomatic and polysymptomatic dogs, cPCR revealed the better performance analysis when compared to other diagnostic methods. The findings presented herein establish cPCR as the most indicated test to detect Leishmania when compared to the other two diagnostic methods evaluated. Despite the fact that the qPCR protocol provides a highly accurate quantification of parasites when targeting the SSU rRNA gene, this technique does not significantly improve the diagnosis of CVL when compared with the performance of the cPCR protocol, which focused on the kinetoplast minicircle.     

Daily rhythms of cortisol and glucose and the influence of the light/dark cycle on anaesthesia in Nile tilapia (Oreochromis niloticus): Does the timing of anaesthetic administration affect the stress response?

Although daily variations in drug pharmacokinetics have been reported for a variety of teleost species, the influence of this daily variation on the cortisol response following anaesthesia remains poorly understood. To address this, two experiments were performed. The first experiment described the daily patterns of cortisol and glucose secretion in tilapia (Oreochromis niloticus). The second experiment investigated how the timing of anaesthetic administration (specifically at mid‐light [ML] or at mid‐dark [MD]) affects the induction and recovery times and plasma cortisol and glucose levels of juvenile Nile tilapia exposed to benzocaine, clove oil or tricaine methanesulphonate (MS‐222). The results revealed that the effect on the stress response associated with the moment when anaesthesia took place (ML or MD) varied according to the treatment (p < 0.05). Cortisol levels were significantly higher at ML for MS‐222 (ML = 116.23 ± 25.55; MD = 48.25 ± 22.33 ng/dl) (p < 0.05) and clove oil (ML 59.73 ± 14.27; MD 38.26 ± 12.07 ng/dl) (p < 0.05), whereas no significant differences were found between ML and MD cortisol levels for the control treatment (ML = 72.91 ± 18.42; MD = 64.80 ± 10.68 ng/dl) (p > 0.05) or in the benzocaine‐treated group (ML = 38.7 ± 4.90; MD = 38.60 ± 3.69 ng/dl) (p > 0.05). The highest plasma cortisol level in ML was found in the MS‐222‐treated group. All the tested anaesthetics had similar cortisol levels at MD (p > 0.05).     

Diagnostic investigation of chronic porcine reproductive and respiratory syndrome virus in a breeding herd of pigs

Forty-five sows and 15 boars were selected at random from a breeding herd known to be chronically infected with porcine reproductive and respiratory syndrome virus (PRRSV) and lymphoid, immune-privileged, and non-lymphoid/non-immune-privileged tissues were tested for the presence of the virus by PCR, virus isolation, and immunohistochemistry. The virus was isolated from the lateral retropharyngeal lymph node of one sow; the isolate was nucleic acid sequenced and determined to be of field origin, and it was inoculated into two PRRSV-naive pregnant sows (A and B) at 95 days of gestation. They were necropsied 14 days later and samples of maternal and fetal tissue and blood samples were collected. Sow A had 10 fresh, six partially autolysed, and two mummified fetuses, and sow B had six fresh and viable fetuses. Viral nucleic acid was detected by PCR in tissue pools from each sow and also from pooled fetal tissues, and the virus was isolated from fetal pools from sow A.     

Immunization with Eimeria ninakohlyakimovae-live attenuated oocysts protect goat kids from clinical coccidiosis

Caprine coccidiosis, affecting mainly young goat kids around the weaning period, is worldwide the most important disease in the goat industry. Control of caprine coccidiosis is increasingly hampered by resistances developed against coccidiostatic drugs leading to an enhanced need for anticoccidial vaccines. In the current study we conducted an oral immunization trial with live attenuated sporulated Eimeria ninakohlyakimovae oocysts. Sporulated E. ninakohlyakimovae oocysts were attenuated by X-irradiation technique. The experimental design included a total of 18 goat kids divided into the following groups: (i) animals immunized with attenuated E. ninakohlyakimovae oocysts at 5 weeks of age and challenged 3 weeks later with non-irradiated homologous oocysts (group 1); (ii) animals infected with non-attenuated E. ninakohlyakimovae oocysts at 5 weeks of age and challenged 3 weeks later with non-attenuated homologous oocysts (group 2); (iii) animals primary-infected with untreated E. ninakohlyakimovae oocysts at 8 weeks of age (control of the challenge infection, group 3); (iv) non-infected control animals (group 4). Goat kids immunized with live attenuated E. ninakohlyakimovae oocysts (group 1) excreted significantly less oocysts in the faeces (95.3% reduction) than kids infected with non-attenuated ones (group 2). Furthermore, immunization with live but attenuated oocysts resulted in ameliorated clinical coccidiosis compared to goat kids infected with untreated oocysts (group 2) and resulted in equally reduced signs of coccidiosis after challenge infection compared to acquired immunity driven by non-attenuated oocysts. Overall, the present study demonstrates for the first time that live attenuated E. ninakohlyakimovae oocysts orally administered showed almost no pathogenicity but enough immunogenicity in terms of immunoprotection. Importantly, vaccinated animals still shed low amounts of oocysts, guaranteeing environmental contamination and consecutive booster infections to sustain ongoing immunity.     

Carotenoid compounds in grapes and their relationship to plant water status

The aim of this work was to study the relationship between carotenoid contents in grapevine berries and plant water status. For this purpose, a black grapevine variety, Vitis vinifera L. cv. Touriga Nacional, was studied. The experiments were carried out in the same Douro vineyards, with plants of the same age, in two different water retention soils. A higher water retention capacity soil, soil A, and a lower water retention capacity soil, soil B, were both in a 1.2 m deep silt-loam schist-derived soil. The training system was the double cordon trained and spur pruned. A first range was nonirrigated (NI) and a second one was irrigated (I), 60% of evapotranspiration (ET(0)). For soil B, a 30% of ET(0) treatment was also applied. The plant water status was estimated by predawn leaf water potential. The effects of plant water status on berry growth were studied by measurement of the berry weight and total soluble solids (degrees Brix). The carotenoid profile was quantitatively determined by high-performance liquid chromatography/diode array. Carotenoids determined were beta-carotene, lutein, neoxanthin, violaxanthin, and luteoxanthin. The comparison between irrigated and nonirrigated grapes was followed from 2 weeks before veraison until the ripe stage. Results showed that at harvest time, berries exposed to the NI had a lower weight than those exposed to the irrigated treatment (60% of ET(0)), 0.89 vs 1.36 g/berry and 0.94 vs 1.34 g/berry, for soils A and B, respectively. The irrigated treatment contributed to a higher sugar concentration in both soils. However, depending on the soil water retention capacity, the carotenoid contents were different in soils A and B. For soil A, the total carotenoid content was similar for both NI and I treatments. However, with regard to soil B, in irrigated treatment, levels of carotenoids were approximately 60% lower than those found for the NI. It seems to be possible to produce higher weight berries (with higher sugar levels) with similar carotenoid contents. On the other hand, soil characteristics had a larger influence than irrigation on the concentration of carotenoids in grapes, resulting in an important viticultural parameter to take into account in aroma precursor formation.