A multiplex PCR for the detection of Brucella spp. and Leptospira spp. DNA from aborted bovine fetuses

Bovine brucellosis and leptospirosis are important causes of bovine abortion around the world. Both diseases can be serologically diagnosed, but many factors may cause false positive and negative results. Direct methods based on bacteriological isolation are usually employed, but they are difficult, time consuming and dangerous. Monoplex polymerase chain reaction (PCR) have been successfully described for the detection of Brucella spp. and Leptospira spp. Aiming at improvement in the direct diagnosis, a multiplex PCR (mPCR) for the detection of these agents in aborted bovine fetuses is described. The detection threshold of the mPCR was evaluated in experimentally contaminated bovine clinical samples using a conventional proteinase K/SDS or a boiling-based extraction protocols. The mPCR was applied to two groups of clinical samples: 63 episodes of bovine abortion and eight hamsters experimentally infected with Leptospira interrogans serovar pomona. Adopting microbiological isolation as reference, the test showed a sensitivity of 100% in both groups of clinical samples. Seven samples collected from bovine fetuses were Brucella spp. culture negative but showed positive results in mPCR. Regarding Leptospira spp. detection, similar results were observed in three bovine clinical samples. All hamsters infected with Leptospira were positive in both microbiological culture and mPCR. The boiling extraction protocol showed better results in some clinical samples, probably by the removal of PCR inhibitors by heat treatment. The high sensitivity, simplicity and the possibility of detection of both bacteria in a single tube reaction support the use of the mPCR described in the routine diagnosis.     

Feed restriction followed by realimentation in prepubescent Zebu females

In the present study, the effect of restricting the feed intake for 77 days and subsequent compensatory growth for 50 days of Sindi females were evaluated. Eighteen animals with an initial age of 21 months and a mean weight of 211.7 kg were placed into three groups according to the following alimentary regimens: feed ad libitum, feed restricted to 20 % dry matter, and feed restricted to 40 % dry matter. In the feed-restriction phase, the nutrient intake decreased (P?<?0.001) with an increase in the restriction level. As a consequence, the observed decrease in ingestion and serum concentrations of total protein, albumin and globulin, urea, glucose, calcium, and phosphorus were inversely proportional (P?<?0.001) to the restriction level. Significant differences in the nutrient intake and serum concentration were not observed in the realimentation phase (P?<?0.05). When animals in the control group reached the end of the feed-restriction phase, their weights (P?<?0.05) were similar to those in the 20 % restricted group, and both obtained a final weight that was greater than that of animals in the 40 % restricted group. In the feed-restriction phase, the control group had a similar mean daily weight gain (P?>?0.05) to animals in the 20 % restricted group and (P?<?0.05) 40 % restricted group. However, in the realimentation phase, the 40 % restricted group obtained greater weight gain rates (P?<?0.05), better food conversions, and partial compensatory gains. In particular, none of the restricted groups reached the final weight of the control group. In the feed-restriction phase, ingested nitrogen, nitrogen excreted in urine and feces, nitrogen balance and retained nitrogen decreased (P?<?0.05) with an increase in the restriction level. In the realimentation phase, none of the nitrogen balance variables were influenced by the restriction level (P?<?0.05). Females in the 40 % restricted group presented better food conversion rates and greater weight gains in the realimentation phase. Based on the animals’ compensatory weight gain, a feed-restriction rate of 20 and 40 % can be adopted as a nutritional management practice for prepubescent Sindi females.     

Interaction between melatonin and follicle-stimulating hormone promotes in vitro development of caprine preantral follicles

The aim of this study was to investigate the effects of melatonin and follicle-stimulating hormone (FSH) on the in vitro culture of goat preantral follicles. Ovarian fragments were cultured for 7 d in α-minimum essential medium (α-MEM⁺) containing melatonin (100, 250, 500, or 1,000 pM), FSH (50 ng/mL), or a combination of the 2 hormones and further analyzed by histology and transmission electron and fluorescent microscopy. The results showed that after 7 d of culture, tissues cultured in α-MEM⁺ alone or supplemented with FSH alone, melatonin (500 and 1,000 pM), or the combination of FSH and melatonin (1,000 pM) maintained percentages of normal preantral follicles similar to the fresh control. In contrast to the noncultured tissues, the percentage of developing follicles was increased under all culture conditions after 7 d (P < 0.05). The addition of 1,000 pM melatonin associated with FSH to the culture medium increased follicular and oocyte diameters compared with α-MEM⁺ alone after 7 d of culture (P < 0.05). Ultrastructural and fluorescent analyses confirmed the integrity of follicles cultured with 1,000 pM of melatonin plus FSH for 7 d. In conclusion, this study demonstrated that the interaction between melatonin and FSH maintains ultrastructural integrity and stimulates further growth of cultured caprine preantral follicles.     

Grafts of porcine small intestinal submucosa seeded with cultured homologous smooth muscle cells for bladder repair in dogs

Background

Due to numerous complications associated to gastrointestinal augmented cystoplasty, this study aimed to analyze the anatomic repair of the bladder of 10 female dogs using grafts of porcine small intestinal submucosa (SIS) seeded with cultured homologous smooth muscle cells, and compare them with the acellular SIS grafts.

Results

We assessed the possible side effects and complications of each type of graft by clinical examination, abdominal ultrasound and laboratory findings. Anatomic repair of neoformed bladder was assessed by histological staining for H/E and Masson''s Trichrome, analyzed with a Nikon Photomicroscope connected to the system of image analysis Image J.

Conclusions

We propose that SIS associated to homologous smooth cells can improve the quality of tissue repair, and consequently decrease the potential complications inherent to acellular SIS.     

Landscape pattern changes over 25 years across a hotspot zone in southern Brazil

Forest fragmentation caused by human activities has many implications for natural landscapes, such as habitat reduction and the loss of biodiversity. This study investigated the temporal fragmentation process of forest remnants in a strongly agro-industrialised region in southern Brazil over 25 years. The studied watershed area hosts two important typologies of the Atlantic Forest biome as well as Quaternary remnants of the Brazilian Savanna biome, which are considered hotspots of biodiversity and reflect the intense process of forest fragmentation caused by Brazilian urban and agro-industrial development. Thus, studies encompassing multitemporal scales are paramount to understanding changes in forest patterns and are fundamental for trend predictions of landscape dynamics. To perform the calculation of the mean normalised difference vegetation index, Landsat satellite images from 1991 to 2016 were processed using SPRING® software. Subsequently, FRAGSTATS® software was used to calculate landscape metrics. A reduction in the number of forest fragments since 1991 was observed, with a maximum amount of 5 243 fragments in 1993 that declined to 4 015 fragments in 2016. Although the number of fragments in the watershed decreased, the mean area increased by 72.9% and the mean of the shape index increased from 1.3 in 1991 to 1.5 in 2016. In addition, there was a 64.7% increase in the edge density and a reduction of 35.6 m in the isolation between the nearest neighbours. The degree of isolation of the fragments underwent a process of expansion and reduction when compared to 1991, presenting results that support the hypothesis that the Atlantic Forest is in a process of stabilisation and forest restoration.     

Identification of fructooligosaccharides in different banana cultivars

Banana has been currently indicated as a good source of fructooligosaccharides (FOS), which are considered to be functional components of foods. However, significant differences in their amounts in bananas have been observed in the literature. This work aims to identify and quantify FOS during ripening in different banana cultivars belonging to the most common genomic groups cultivated in Brazil. Considering that these differences can be due to cultivar, stage of ripening, and the methodologies used for FOS analyses, sugar contents were analyzed by high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD) and gas chromatography-mass spectrometry (GC-MS). An initial screening of eight cultivars (Ouro, Nanic?o, Prata, Ma??, Mysore, Pacovan, Terra, and Figo) in a full-ripe stage showed that 1-kestose, the first member of the FOS series (amounts between 297 and 1600 microg/g of DM), was accumulated in all of them. Nystose, the second member, was detected only in Prata cultivar. Five of the cultivars were analyzed during ripening, and a strong correlation could be established with a specific sucrose level ( approximately 200 mg/g of DM), which seems to trigger the synthesis of 1-kestose (the low amounts of FOS, below the functional recommended dose, indicates that banana cannot be considered a good source of FOS).     

Triploid Induction in the Yellowtail Tetra,Astyanax altiparanae,Using Temperature Shock: Tools for Conservation and Aquaculture

Triploidization is an interesting tool to produce sterile fish. In the yellowtail tetra, Astyanax altiparanae, this can be applied for aquaculture and surrogate technologies. In this study, we compared the efficacy of cold (2 C) or heat shock (38 C, 40 C, and 42 C) on triploid induction in the yellowtail tetra. The eggs were treated with cold or heat shock, 2 min postfertilization (30 min in cold shock or 2 min in heat shock). Intact embryos served as the control group. Ploidy status was confirmed by karyotyping, flow cytometry, and nuclear diameter of erythrocytes. The hatching rate decreased after cold shock (12.69 ± 15.76%) and heat shock at 42 C (0.35 ± 0.69%) in comparison with the control group (63.19 ± 16.82%). At 38 C and 40 C, hatching rates (61.29 ± 17.73% and 61.75 ± 22.1%, respectively) were not decreased. Only one triploid arose at 38 C (1/80). At 40 C, a high number of triploids arose (72/78). At 42 C, very few embryos developed into the hatching stage. A large number of haploid individuals arose after cold shock (61/75), with only one triploid. Our results indicate that heat shocking of embryos at 40 C is optimum for triploid production in the yellowtail tetra.     

Factors affecting width of the canine femorotibial joint space in nonweight-bearing radiographs.

Caudocranial stifle radiographs with variations in positioning were made in two greyhound cadavers. Radiographs were repeated after each of three interventions: cranial cruciate desmotomy; release of the caudal horn of the medial meniscus; complete medial meniscectomy. The joint space on medial and lateral aspects of the joint was measured by a observer who was unaware of positioning or intervention. One dog had significantly wider joint space than the other (1.0 vs. 1.5mm). The lateral aspect of the joint space was wider than the medial aspect (1.7 vs. 0.7 mm). Medial rotation of the stifle resulted in an increase of 0.4 mm in width of the lateral joint space, whereas lateral rotation of the stifle reduced the lateral joint space by an average of 0.4 mm. Decentering the X-ray beam had no significant effect on joint space width. Tension increased the width of the medial joint space by an average of 1.2 mm and the lateral aspect by an average of 1.3 mm. Cranial cruciate desmotomy resulted in an average 0.3 mm increase in width of the joint space, and medial meniscectomy with an average 0.2 mm reduction of the joint space. Although the femorotibial joint space was affected by iatrogenic stifle injuries and by medial or lateral rotation, these changes were less than the differences between the two dogs. Hence it seems unlikely that the small changes in joint space width associated with cruciate ligament desmotomy and medial meniscectomy will be detectable in clinical practice.     

Quantification of EGFR family in canine mammary ductal carcinomas in situ: implications on the histological graduation

Veterinary Research Communications - The epithelial growth factor receptors are transmembrane proteins with an important role in the neoplastic progression of tumors, and in this context, DCIS is...     

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.