Design and validation of a RT‐qPCR procedure for diagnosis and quantification of most types of infectious pancreatic necrosis virus using a single pair of degenerated primers

Infectious pancreatic necrosis virus (IPNV) is an important virus which affects the salmonid aquaculture industry worldwide; therefore, it is important to develop rapid and reliable methods of diagnosis to detect the disease at early stages. Nowadays, RT‐qPCR is replacing other methods because it provides additional information on the viral load, which is important to have a better understanding of the virus replication level and of the stage of the infection and its risk level. The main problem stems from the high diversity of this virus, which can compromise the reliability of the diagnosis. In this study, we have designed an RT‐qPCR procedure for diagnosis and quantification of IPNV based on a single pair of primers targeted to segment B. The procedure has been validated, in vitro and in vivo, testing two different types of standards against seven reference strains and 23 field isolates from different types. The procedure is reliable for the detection of any type, with a detection limit of 31 TCID₅₀ mL^?1, 50 pfu mL^?1 or 66 RNA copies mL^?1, depending on the standard. All the standard curves showed high reliability (R² > 0.95). The results support the high reliability of this new procedure for the diagnosis and quantification of IPNV.     

Black carbon estimation in French calcareous soils using chemo‐thermal oxidation method

We studied the black carbon (BC) content of ca. 405 samples from French topsoil and artificial soil and carbonate mixtures. Our protocol involved three main steps: (i) decarbonation by HCl, (ii) elimination of non‐pyrogenic organic carbon in a furnace at 375 °C, and (iii) quantification of residual carbon by CHN analysis. BC content increased for calcareous soils according to their carbonates content. Subsequent analyses confirmed the existence of a methodological artefact for BC determination only in calcareous soils. Decarbonation changes the thermal properties of organic matter, creating more recalcitrant carbon than in the initial sample. Higher CaCO₃ and organic carbon content results in a more pronounced artefact. The reversal of the first two steps of the chemo‐thermal oxidation method (i.e. thermal oxidation before soil decarbonation) eliminates this artefact. Overall, our results suggest that BC content may have been overestimated in a large number of studies on calcareous soils.     

Comparative Characterization of Enzymatic Digestion from Fish and Soybean Meal from Simulated Digestive Process of Pacific Bluefin Tuna,Thunnus orientalis

The digestive process of the Pacific bluefin tuna (PBT), Thunnus orientalis, was simulated through two phases of in vitro digestion: acidic digestion with porcine pepsin, followed by alkaline digestion with pancreatic crude extract (PCE) obtained from the PBT to hydrolyze fish meal (FM) and soybean meal (SBM) as protein substrates. The crude protein from FM resulted in a lower degree of hydrolysis (73.3%) compared with SBM (79.2%). However, the resulting digested products showed that FM contained 35% more small peptides, with sizes <6.5 kDa than those from the starting material (>150 kDa). The SBM had an increase of only 1.3% in the similar peptide cut‐offs found after hydrolysis. These results suggested that FM appeared to be a better source of protein according to the amount of low‐molecular weight peptides. In addition, the proteolytic activity of PCE showed that 88.9% of its alkaline proteolytic activity corresponded to trypsin and 2.9% corresponded to chymotrypsin activity. The results shown here demonstrate that peptide sizes are important in identifying suitable protein sources for aquafeed production to reinforce the primary results obtained from the in vitro digestibility using the pH‐Stat system. These results also contribute to a better understanding of the digestibility process in aquatic organisms.     

Origin and distribution of facial nerve anatomy in van cats

This study aims to reveal the morphological properties of facial nerve and the middle ear in Van cats. Study material was composed of 6 female Van cats. Dissections were performed under a Zoom Stereo Microscope. There was no plexus buccalis in Van cats. The chorda tympani was observed to pass through an opening in the tympanic cavity, emerge through a small opening just behind the retroarticular process, and join the lingual nerve. A rounded anatomical formation with a size of 2.75 ± 0.3 mm was found to be located within the mastoid process of the temporal bone between the facial nerve and the auricular branch of the vagus nerve. The stapes nerve was not present. The geniculate ganglion was very prominent and about 1.00 mm high. The deep petrosal nerve was observed to emerge from the plexus tympanicus. The bulla tympanica was 18.96 ± 0.10 mm long, 13.03 ± 0.20 mm wide and 13.16 ± 0.20 mm high. After leaving the mandibular nerve, the n.tensoris tympani coursed caudally around the a.maxillaris, formed an ansa, entered the tympanic cavity through the canalis musculotubarius and reached an end in the m. tensor tympani. Due to the scarcity of studies on the middle ears of Van cats, it is thought that this study will fill a gap in the field of veterinary anatomy.     

Use of dimethylformamide to cryopreserve alpaca semen previously incubated with collagenase

The objective of this study was to evaluate the effect of collagenase and two final dimethylformamide (DMF) concentrations (4% and 7%) on alpaca frozen-thawed sperm quality. A total of 25 ejaculates from 5 alpaca were obtained using electroejaculation. Each individual ejaculate was evaluated and then diluted 4:1 in a solution of 1 mg/ml collagenase in HEPES-TALP medium and incubated for 4 min at 37°C. Subsequently, samples were diluted in TRIS-fructose-citric acid-egg yolk and cooled to 5°C. Then, each sample was divided in two aliquots and DMF at final concentration of 4% or 7% was added, equilibrated for 1 hr at 5°C and frozen over liquid nitrogen vapours. A Kruskal–Wallis test was used to evaluate the sperm morphometry, and Completely Random Block designs were used to analyse sperm motility, viability, membrane function and acrosome status. After collagenase incubation, none of the samples showed thread formation, and sperm parameters were preserved. Non-progressive motile sperm were higher (p < .05) in equilibrated samples (4% DMF: 31.8 ± 8.3% and 7% DMF: 36.3 ± 11.8%) compared to raw (10.1 ± 4.3%) and frozen-thawed semen (4% DMF: 9.7 ± 1.8% and 7% DMF: 7.5 ± 3.2%). Sperm membrane function, membrane integrity and intact acrosomes were higher (p < .05) in raw semen (40.1 ± 12.2%, 94.6 ± 3.2% and 91.3 ± 8.1%) compared to frozen-thawed samples (4% DMF: 19.8 ± 4.7%, 53.2 ± 2.7%, 65.7 ± 8.7% and 7% DMF: 20.4 ± 4.5%, 54.1 ± 1.4%, 64.6 ± 9.1%). Length of the sperm head was lower in frozen-thawed samples, being statistically different with 4% DMF compared to pre-freezing samples. The ratio between acrosome and head areas was greater (p < .05) in frozen-thawed samples. Incubation of raw alpaca semen with collagenase decreased the thread formation without affecting sperm quality. Frozen of collagenase treated alpaca semen with 4% or 7% DMF did not preserve the sperm parameters in thawed samples.