Optimizing injection time of GFP plasmid into sheep zygote

Microinjection of exogenous DNA into the cytoplasm of matured oocytes or zygotes is a promising technique to generate transgenic animals. However, the data about the microinjection time and procedure in sheep are limited and have not treated in detail. To obtain more in-depth information, the Sarda sheep oocytes from abattoir-derived ovaries were subjected to IVM and IVF. Then, the GFP plasmid as a reporter gene was injected into the cytoplasm of MII oocytes (n: 95) and zygotes at different post-insemination intervals (6–8 hpi, n: 120; 8–10 hpi, n: 122; 10–12 hpi, n: 110 and 12–14 hpi, n: 96). There were no significant differences in the cleavage rates between the groups. However, blastocyst rate of injected zygotes at all-time intervals was significantly lower than injected MII oocytes and control group (p < 0.05). Interestingly, the proportion of GFP-positive embryos was higher at 8–10 hpi compared with other injected groups (4 % versus 0 %, p  < 0.01). Among these, the proportion of mosaic embryos was high and two of those embryos developed to the blastocyst stage. In conclusion, we settled on the cytoplasmic microinjection of GFP plasmid at 8–10 hpi as an optimized time point for the production of transgenic sheep and subsequent experiments.     

Molecular characterisation of Theileria equi and risk factors associated with the occurrence of theileriosis in horses of Punjab (Pakistan)

Theileria equi (T. equi) is an obligate intra- and extra-erythrocytic parasite that causes equine theileriosis (ET) in equids. Equine theileriosis is considered a notifiable disease of global significance, a major constraint to the international movement of horses, and endemic in many countries. This disease may be difficult to diagnose, as it can produce variable and nonspecific clinical signs. A cross-sectional study was designed for the molecular characterisation of T. equi and to investigate the associated risk factors of ET accompanied by its consequences on haematological and sero-biochemical parameters. A convenience sampling of 500 blood samples were collected from ET suspect horses from January to December 2017. PCR was performed on all blood samples targeting the 18S rRNA gene of T. equi followed by sequencing; 9% animals tested positive with confirmed sequences. The isolates of this study showed high homology with Cuban, Russian and Brazilian isolates of T. equi (accession numbers KY111762.2 , MG551915.1 and KY952237.1 , respectively). Based on multivariate analysis, the principal risk factors consisted of absence of dogs on the premises and presence of tick infestation. The haemato-biochemical parameters showed a decrease in granulocytes and erythrocytes, and an increase in lymphocytes, monocytes, mean corpuscular volume, mean corpuscular haemoglobin, mean platelet volume, glucose, phosphorus and aspartate aminotransferase in positive horses. This is the first study which identified ET in Punjab (Pakistan) using molecular techniques and risk factors together with the haemato-biochemical variations in horses.     

The effect of dry conditions on subsequent leakage and rotting of fungal sclerotia

Dried sclerotia of Sclerotium delphinii rotted in moist soil whereas those of Sclerotium cepivorum. Botrytis cinerea and Botrytis tulipae did not. A number of fungi invaded dried sclerotia of S. delphinii in soil, the principal coloniser found in the first sampling being Trichodermu hamatum. Leakage of ¹⁴C compounds from dried labelled sclerotia placed in water was rapid and was little affected by variation in leakage temperature from 1 to 25°C or by prolonging the drying period beyond a day. Leakage from dried sclerotia which were allowed to imbibe water through a small part of their surface was much reduced. Sclerotia which were redried after leakage leaked again when returned to water but with all four fungi the first of three leakage cycles gave the highest ¹⁴C levels. Loss in dry weight in the first leakage cycle was greater with S. delphinii than with the other three fungi and this may be related to the poor survival of dried sclerotia of S. delphinii in moist soil. Substances lost during leakage appear to originate from within sclerotial hyphae rather than from the hyphal free space.     

Effect of IAA and GA3 on the Anatomical Characteristics, Straw and Fiber Yield and Quality of Flax

IAA and GA₃ had increased the stem thickness by increasing that of cortex, phloem fibers and xylem. Each had increased straw and fiber yield/plant, but the effect was more pronounced with GA₃ than IAA. The percentage of long fibers, strength and fineness increased significantly with GA₃. The latter two properties decreased with IAA.     

Polyethylene mulching of soil to reduce viability of sclerotia of Sclerotium oryzae

Mulching of Sclerotium oryzae infested soil (moist or dry) with polyethylene sheets during hot summer days of May and June increased the soil temperature at 5 cm from 36°C (unmulched) to 48°C (wet) and from 44 to 52°C (dry) and at 20cm from 32 to 38°C (wet) and from 35 to 39°C (dry). In artificially-infested soil, the sclerotia were not eradicated but 95–100% loss in viability was observed at 5 cm by a mulch treatment for 1 week and at 20 cm by mulching for 8 weeks. Mulching effects were not influenced by moisture content of soil or by amendments with lucerne or wheat straw. Mulching of naturally-infested soil at a second site did not eradicate S. oryzae but reduced sclerotial viability by 93%.     

Effect of Bacillus thuringiensis var. kurstaki on survival and mortality of immature and mature stages of Diadegma insulare parasitizing Plutella xylostella

Effects of Bacillus thuringiensis (Bt) on immature stages of Diadegma insulare (Cresson) (Hym., Ichneumonidae) within larvae of diamondback moth (DBM), Plutella xylostella (L.) (Lep., Plutellidae), and on the adult stage as a Bt-honey solution were evaluated at 25?±?1?°C, 65?±?10% r.h. and 16:8?h (L:D) photoperiod. P. xylostella and D. insulare pupae were collected from cabbage fields in Karaj, Iran. A commercial wettable powder formulation of Bt (var. kurstaki, serotype H-3a3b, strain Z-52) was used in these experiments. The value of LC₅₀ for third instar larvae of P. xylostella was 210?ppm. Parasitoid??s adult mortality at field rate of Bt was not significantly different from that of control. Formation of parasitoid??s pupae in Bt-parasitoid combined treatments was significantly lower than parasitoid alone. Formation of parasitoid??s pupae in larvae treated with LC₁₀, LC₃₀ and LC₅₀ of Bt and then exposed to parasitoids after 48?h was 5%, 3% and 2%, respectively, which was significantly lower than that of the parasitoid alone (41%). Mean percentage mortality of larvae of DBM in LC₃₀ and LC₅₀ concentrations of Bt combined with the parasitoid was approximately 100%. Our results showed that B. thuringiensis kills D. insulare larvae indirectly by killing susceptible hosts in which they are developing. Successful use of multiple biological control agents will depend on close monitoring of their compatibility in the field. Our results could be useful in this purpose.     

Uptake and remobilization of selenium in Brassica napus L. plants supplied with selenate or selenium‐enriched plant residues

Selenium (Se) biofortification via crops is one of the best strategies to elevate the daily Se intake in areas where soil Se levels are low. However, Se fertilizer recovery (SeFR) is low and most of the Se taken up accumulates in non‐harvested plant parts and returns to the soil with plant residues. A pot experiment with soil was undertaken to study the efficiency of inorganic Se (Na₂SeO₄) and Se‐enriched plant residues for biofortification, as well as to identify the bottlenecks in Se utilization by Brassica napus L. The soil was fertilized with Na₂SeO₄ (0 and 7 µg Se kg^?1) or with Se in stem or leaf residues (0 and 7 µg Se kg^?1). A treatment with autoclaved soil was included (0 and 7 µg kg^?1 as Na₂SeO₄) to unravel the impact of microbial activity on Se uptake. The Se‐enriched plant residues produced a lower Se uptake efficiency (SeUPE) and SeFR than did inorganic Se, and soil autoclaving enhanced Se accumulation in the plants. The time required for decomposition seems to preclude crop residues as an alternative source of Se. Furthermore, B. napus had a limited capacity to accumulate Se in seeds. The study shows that the bottlenecks in Se biofortification appear to be its low bioavailability in soil and poor loading from the silique walls to seeds. Thus, improved Se translocation to seeds would be a useful breeding goal in B. napus to increase SeFR.     

The first report on serotyping of bluetongue virus in small ruminants of Khyber Pakhtunkhwa province,Pakistan

Bluetongue virus (BTV), a member of Orbivirus genus (family Reoviridae), is a non-contagious infection of domestic and wild ruminants. The current study was designed to detect various serotypes of BTV in small ruminants of Khyber Pakhtunkhwa (KPK) province of Pakistan, along with their effects on hemato-biochemical parameters. A total of 408 serum samples in four districts (Mansehra, Abbottabad, Swabi, and Kohat) of KPK from small ruminants were screened based on competitive ELISA (cELISA). A total of 204 (50%) samples were found positive for BTV group–specific antibodies. The seropositive samples were processed for the detection of BTV serotypes through real-time polymerase chain reaction (qPCR). Out of 204 cELISA-positive samples, 60 (29.41%) were found positive through qPCR. Three serotypes [6, 8, 9] were detected from Mansehra District and two from Kohat [2, 8] and Abbottabad [6, 8], while only one from Swabi [8]. The serotype “8” was found consistently in all the four study districts. A significant (p?<?0.05) increase in the level of blood urea nitrogen (BUN) and alkaline phosphatase (ALP) was recorded in goats, whereas aspartate aminotransferase (AST) in sheep infected with BTV, compared to healthy animals. The hematological parameters showed significantly (p?<?0.05) raised total leucocyte count (TLC) in both sheep and goats, whereas only hematocrit (HCT) value was increased significantly (p?<?0.05) in infected sheep. This is the first report on serotyping of BTV among small ruminants in Pakistan.

     

Integrated approach for improving small scale market oriented dairy systems in Pakistan: participatory rural appraisal and economic opportunity survey

Livestock production is an integral part of the rain-fed and irrigated agriculture system in Pakistan. Animal production is closely interlinked with the cropping systems and play a crucial role in the rural economy. Participatory rural appraisals and economic opportunity surveys were conducted in two ecological zones (irrigated and rain-fed) and two dairy production systems (peri-urban and mixed livestock). Major constraints in animal health, nutrition and reproduction were identified and interventions were suggested to overcome these difficulties. The economic opportunity survey revealed that maximum opportunity to enhance farmers’ income is to increase milk production per day per animal, which can be accomplished through coordinated improvements in nutrition, reproduction and genetics.     

A Bayesian Generalized Linear Model for Crimean–Congo Hemorrhagic Fever Incidents

Global spread of the Crimean–Congo hemorrhagic fever (CCHF) is a fatal viral infection disease found in parts of Africa, Asia, Eastern Europe and Middle East, with a fatality rate of up to 30%. A timely prediction of the prevalence of CCHF incidents is highly desirable, while CCHF incidents often exhibit nonlinearity in both temporal and spatial features. However, the modeling of discrete incidents is not trivial. Moreover, the CCHF incidents are monthly observed in a long period and take a nonlinear pattern over a region at each time point. Hence, the estimation and the data assimilation for incidents require extensive computations. In this paper, using the data augmentation with latent variables, we propose to utilize a dynamically weighted particle filter to take advantage of its population controlling feature in data assimilation. We apply our approach in an analysis of monthly CCHF incidents data collected in Turkey between 2004 and 2012. The results indicate that CCHF incidents are higher at Northern Central Turkey during summer and that some beforehand interventions to stop the propagation are recommendable. Supplementary materials accompanying this paper appear on-line.