Effect of Physiologically Relevant Heat Shock on Development,Apoptosis and Expression of Some Genes in Buffalo (Bubalus bubalis) Embryos Produced In Vitro

For investigating the effects of physiologically relevant heat shock, buffalo oocytes/embryos were cultured at 38.5°C (control) or were exposed to 39.5°C (Group II) or 40.5°C (Group III) for 2 h once every day throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC). Percentage of oocytes that developed to 8‐cell, 16‐cell or blastocyst stage was lower (p < 0.05) and the number of apoptotic nuclei was higher (p < 0.05) for Group III > Group II > controls. At both 8–16‐cell and blastocyst stages, relative mRNA abundance of stress‐related genes HSP 70.1 and HSP 70.2 and pro‐apoptotic genes CASPASE‐3, BID and BAX was higher (p < 0.05) in Groups III and II than that in controls with the exception of stress‐related gene HSF1. Expression level of anti‐apoptotic genes BCL‐XL and MCL‐1 was also higher (p < 0.05) in Groups III and II than that in controls at both 8–16‐cell and blastocyst stages. Among the genes related to embryonic development, at 8–16‐cell stage, the expression level of GDF9 was higher (p < 0.05) in Group III than that in controls, whereas that of GLUT1, ZAR1 and BMP15 was not significantly different among the three groups. At the blastocyst stage, relative mRNA abundance of GLUT1 and GDF9 was higher (p < 0.05) in Group II than that in controls, whereas that of ZAR‐1 and BMP15 was not affected. The results of this study demonstrate that exposure of buffalo oocytes and embryos to elevated temperatures for duration of time that is physiologically relevant severely compromises their developmental competence, increases apoptosis and affects stress‐, apoptosis‐ and development‐related genes.     

Buffalo (Bubalus bubalis) ES Cell–Like Cells are Capable of In Vitro Skeletal Myogenic Differentiation

When buffalo embryonic stem (ES) cell–like cells that expressed surface markers SSEA‐4, TRA‐1‐60, TRA‐1‐81, CD9 and CD90 and intracellular markers OCT4, SOX2 and FOXD3, as shown by immunofluorescence, and that expressed REX‐1 and NUCLEOSTEMIN as confirmed by RT‐PCR, were subjected to suspension culture in hanging drops in absence of LIF and buffalo foetal fibroblast feeder layer support, they differentiated to form three‐dimensional embryoid bodies (EBs). Of 231 EBs examined on Day 3 of suspension culture, 141 (61.3 ± 3.09%) were of compact type, whereas 90 (38.4 ± 3.12%) were of cystic type. The cells obtained from EBs were found to express NF‐68 and NESTIN (ectodermal lineage), BMP‐4 and α‐skeletal actin (mesodermal lineage), and α‐fetoprotein, GATA‐4 and HNF‐4 (endodermal lineage). When these EBs were cultured on gelatin‐coated dishes, they spontaneously differentiated to several cell types such as epithelial‐ and neuron‐like cells. When EBs were cultured in the presence of 1 or 2% DMSO or 10^?8 m or 10^?7 m retinoic acid for 25 days, ES cells could be directed to form muscle cell–like cells, the identity of which was confirmed by expression of α‐actinin by immunofluorescence and of MYF‐5, MYOD and MYOGENIN genes by RT‐PCR. MYOD was first detected on Day 10 in both treatment groups and on Day 15 in controls, whereas MYOGENIN was first detected on Day 10, Day 15 and Day 25 in the presence of retinoic acid, in the presence of DMSO and in controls, respectively. The present study demonstrates the ability of buffalo ES cell–like cells to undergo directed differentiation to cells of skeletal myogenic lineage.     

Quantitative risk assessment for the annual risk of exposure to Trichinella spiralis in imported chilled pork meat from New Zealand to Singapore

Abstract

AIMS: To determine the annual likelihood of exposure to an infectious dose of Trichinella spiralis from consuming imported pork meat from New Zealand to Singapore.

METHODS: Input values specific for chilled pork meat imported into Singapore from New Zealand were used in a quantitative risk-assessment model. The model, designed to allow any combination of importing and exporting countries, was divided into two components, viz the release assessment, and the exposure assessment that assessed the annual risk of exposure to the consumer (ARC). The former estimated the likelihood that a contaminated fresh meat product from New Zealand would arrive at Singapore's border, and took into consideration the prevalence of disease on different types of farms. The latter determined the likelihood over a year that a person in Singapore would consume one or more servings of imported fresh meat from New Zealand that contained a burden of greater than or equal to one larva(e) of T. spiralis per gram after preparation for consumption.

RESULTS: The ARC for offal was 2.41 × 10^?7, which was below the pre-selected safety threshold of 1.00 × 10^?6. The ARC for lean meat was 2.39 x 10^?5, which was above the acceptable safety threshold.

CONCLUSIONS: The study demonstrated that continued routine testing at slaughter is unnecessary for pig offal produced commercially, and provided a model with which to further assess management of the risk of exposure to T. spiralis in lean meat.

CLINICAL RELEVANCE: The potential of Trichinella species to cause disease in humans is a public health concern, and has created adverse effects on the international trade of fresh lean meat without regard to the surveillance measures employed by particular pork-producing countries.     

Effects of selenium as a beneficial element on growth and photosynthetic attributes of greenhouse cucumber

Selenium (Se) is an essential element for human and livestock with antioxidant and anticancer characteristics. Although Se is not an essential element for plants, it has been reported that it can improve plant growth. This experiment was conducted at the Isfahan University of Technology in winter 2010. The experiment was factorial based on a completely randomized design (CRD) with four replications. Se was added to nutrient solution in four concentrations 2, 4, and 6 mg/L sodium selenite (Na₂SeO₃). Root volume, fresh and dry weights of shoots and roots, number and weight of fruits, chlorophyll content, and photosynthesis traits [photosynthesis rate, stomata internal carbon dioxide (CO₂) concentration, stomata conductance] were measured. Results showed that Se increased root dry weight. Fresh and dry weights of shoot increased in the 2 mg/L Se treatment and decreased at the higher level of Se. Chlorophyll content and photosynthesis rate were not affected by Se. Stomata internal CO₂ concentration and stomata conductance decreased by Se addition. Overall, Se at 2 mg/L application rate was effective in some physiological characteristics of cucumber.     

Effects of mycorrhiza inoculation on cucumber growth irrigated with saline water

This experiment was conducted as a factorial based on complete randomized design (CRD) to study the effects of mycorrhiza inoculation density on cucumber cv. Super N3 irrigated with different salinity sodium chloride (NaCl) levels. Treatments were mycorrhiza inoculations; M1 (non mycorrhizal plant), M2 and M3 (mycorrhiza inoculations with 1000 and 2000 spores) and saline water was provided by S1, S2, S3, and S4 (control, 50, 75, and 100 mM NaCl) with 4 replications. The results showed that saline water reduced root, shoot, and fruit weights, and increased proline and electrolyte leakage. Photosynthesis rate, stomata and mesophyll conductance significantly decreased with increased NaCl concentrations. Mycorrhiza inoculation with 2000 spores increased fruit fresh and dry weights, proline and electrolyte leakage, and both mycorrhiza inoculations increased root and shoot dry weights, photosynthesis and stomata conductance. Root volume increased by mycorrhiza inoculation with 2000 spores under non-stress condition, and root length was stimulated by both mycorrhiza inoculations at all saline water levels. Fruit fresh and dry weights were enhanced by mycorrhiza inoculation with 1000 spores at all saline water levels. Photosynthesis rate was reduced by saline water stress and mycorrhiza inoculation stimulated photosynthesis rate. Mycorrhiza inoculation with 2000 spores increased transpiration under saline and non-saline conditions. Proline content of cucumber leaves increased under saline water application. Electrolyte leakage increased by saline water and mycorrhiza inoculation could not improve it. Both mycorrhiza inoculations (1000 and 2000 spores) at all salinity levels, and mycorrhiza inoculation with 1000 spores at 100 mM NaCl enhanced fruit weight, photosynthesis, and proline content of the cucumber leaves.     

Short‐Term Antiandrogen Flutamide Treatment Causes Structural Alterations in Somatic Cells Associated with Premature Detachment of Spermatids in the Testis of Pubertal and Adult Guinea Pigs

In spite of widespread application of flutamide in the endocrine therapies of young and adult patients, the side effects of this antiandrogen on spermatogenesis and germ‐cell morphology remain unclear. This study evaluates the short‐term androgen blockage effect induced by the administration of flutamide to the testes of pubertal (30‐day old) and adult (65‐ and 135‐day old) guinea pigs, with an emphasis on ultrastructural alterations of main cell types. The testes removed after 10 days of treatment with either a non‐steroidal antiandrogen, flutamide (10 mg/kg of body weight) or a pharmacological vehicle alone were processed for histological, quantitative and ultrastructural analysis. In pubertal animals, flutamide androgenic blockage induces spermatogonial differentiation and accelerates testes maturation, causing degeneration and detachment of primary spermatocytes and round spermatids, which are subsequently found in great quantities in the epididymis caput. In post‐pubertal and adult guinea pigs, in addition to causing germ‐cell degeneration, especially in primary spermatocytes, and leading to the premature detachment of spherical spermatids, the antiandrogen treatment increased the relative volume of Leydig cells. In addition, ultrastructural evaluation indicated that irrespective of age antiandrogen treatment causes an increase in frequency of organelles involved with steroid hormone synthesis in the Leydig cells and a dramatic accumulation of myelin figures in their cytoplasm and, to a larger degree, in Sertoli cells. In conclusion, the transient exposition of the guinea pigs to flutamide, at all postnatal ages causes some degenerative lesions including severe premature detachment of spermatids and accumulation of myelin bodies in Leydig and Sertoli cells, compromising, at least temporarily, the spermatogenesis.     

Assessment of DNA Damage during In Vitro Development of Buffalo (Bubalus bubalis) Embryos: Effect of Cysteamine

Comet assay was used in the present study to examine DNA damage to buffalo oocytes and embryos during in vitro culture. Embryos were produced in vitro from oocytes obtained from slaughterhouse ovaries in presence of cysteamine (IVM and IVC media supplemented with 50 and 100 μm , respectively) or in its absence (controls). Compared to controls, cysteamine supplementation increased (p < 0.01) cleavage rate and proportion of oocytes that developed to 8‐ to 16‐cell stage. The incidence of DNA damage was lower (p < 0.01) in cysteamine group than that in controls at 8‐ to 16‐ (19.3 ± 4.24 vs 72.0 ± 5.22%) but not in 2‐cell stage embryos (11.7 ± 5.63 vs 20.8 ± 5.49%) or in mature oocytes (5.3 ± 3.43 vs 10.3 ± 4.73%). The tail length, which indicates magnitude of DNA damage, was shorter (p < 0.01) in cysteamine group than in controls in mature oocytes (25.5 ± 0.5 vs 36.0 ± 0.71 pixels) and 8‐ to 16‐cell stage (49.2 ± 1.64 vs 152.7 ± 1.28 pixels) but not in 2‐cell stage embryos (36.3 ± 1.54 vs 36.4 ± 0.75 pixels). Also, exposure of oocytes/embryos to UV radiation or H₂O₂ caused extensive DNA damage. In conclusion, these results suggest that oocytes/embryos suffer from DNA damage during progress of in vitro culture, which can be partly ameliorated by cysteamine supplementation of culture media.     

Different Morphologies of Leishmania major Amastigotes with No Molecular Diversity in a Neglected Endemic Area of Zoonotic Cutaneous Leishmaniasis in Iran

Background:

Molecular diversity of Leishmania major and its morphological changes have become a controversial issue among researchers. Some aspects of polymorphic shapes of amastigotes in clinical manifestations along with molecular variation were evaluated among suspected patients of some exceptional zoonotic cutaneous leishmaniasis locations in Northern Khuzestan, Southwestern Iran.

Methods:

Suspected patients (n = 165) were sampled in zoonotic cutaneous leishmaniasis foci over two consecutive years during 2012-2014. Prepared smears were stained, scaled and measured by ocular micrometer. DNA was extracted from smears; ITS-rDNA and Cytochrome b (Cyt b) markers were amplified, and PCR products were digested by BsuR1 restriction enzyme. Then the RFLP and sequencing were employed.

Results:

Only L. major was identified in patients containing regular amastigotes'' shapes (oval or round) with a size of 2-4 µm in each of classical wet, dry, mixed lesions. Meanwhile, irregular shapes (spindle, pear, or cigarette) were observed separately in non-classical wet lesions with more than 4 µm. Interestingly, a few amastigotes with an external flagellum were observed in some lesions. All sequenced ITS-rDNA and Cyt b genes of L. major did not show any molecular variation (χ ² P > 0.05), including only one common haplotype (GenBank access no. {"type":"entrez-nucleotide","attrs":{"text":"EF413075","term_id":"151936076","term_text":"EF413075"}}EF413075).

Conclusion:

Findings proved that unlike other endemic foci, there is not a meaningful correlation between phenotypic and genotypic features of L. major isolates. This study is considered as the first comprehensive report to incriminate morphometric shapes of L. major amastigotes, which enhances our knowledge concerning their relevance with various clinical appearances and genotypic traits. Key Words: Leishmania major, Nuclear gene, Mitochondrial gene, Amastigote shapes, Iran     

Distribution and variability of n-alkanes in waxes of conifers

Epicuticular waxes have vital roles in the growth and development of plants and in defense. Conifers have a considerable amount of waxes on their cones and leaves.Here we characterized the n-alkane composition of Iranian conifers, including Juniperus oblonga, J. foetidissima, J.sabina, J. communis subsp. hemisphaerica, J. excelsa,Cupressus sempervirens, Platycladus orientalis from Cupressaceae and Taxus baccata from Taxaceae for the first time using GC-FID analyses. In the waxes, 25 n-alkane homologs with chain lengths ranging from C7 to C32 were identified. Short-chain n-alkanes were dominant in almost all samples with some exceptions. Complementary studies to elucidate complete wax constituents of Iranian conifers and n-alkane distribution pattern as a function of geographical and bioclimatic variables are recommended.     

Off-axis crustal thickness across and along the east pacific rise within the MELT area

Wide-angle seismic data along the Mantle Electromagnetic and Tomography (MELT) arrays show that the thickness of 0.5- to 1. 5-million-year-old crust of the Nazca Plate is not resolvably different from that of the Pacific Plate, despite an asymmetry in depth and gravity across this portion of the East Pacific Rise. Crustal thickness on similarly aged crust on the Nazca plate near a magmatically robust part of the East Pacific Rise at 17 degrees15'S is slightly thinner (5.1 to 5.7 kilometers) than at the 15 degrees55'S overlapping spreading center (5.8 to 6.3 kilometers). This small north-south off-axis crustal thickness difference may reflect along-axis temporal variations in magma supply, whereas the across-axis asymmetry in depth and gravity must be caused by density variations in the underlying mantle.