Infiltrating Foxp3+ Regulatory T cells and Histopathological Features in Canine Classical and Spermatocytic Seminomas

In humans, regulatory T (T reg) cells are known to play a critical role in both the regulation of immune homoeostasis and the progression of cancer. However, there is little information about the identification, characterization and the function of T reg cells in canine tumours. We identified T reg cells in 28 canine seminoma samples using a Forkhead box P3 (Foxp3) antibody and investigated the relationship between T reg cell infiltration and histopathological features of classical and spermatocytic seminomas (SE and SS, respectively). The Foxp3 protein showed nuclear immunostaining in infiltrating lymphocytes, and Foxp3+ cells were diffused or focally distributed in seminoma tissues. Foxp3+ cells were frequently present in the SS histotype, in seminomas that showed no evidence of tumour cell invasion into the vessels and in seminomas showing a diffuse growth pattern with three cell types. Neither the SE/SS histotype nor the histopathological features of the tumour correlated with Foxp3+ cell counts. These results indicate that Foxp3+ T reg cells may be associated with a less malignant histological phenotype or may not play a critical role in the immune response of canine seminomas. Moreover, Foxp3+ T reg cells may be associated with SS seminoma, but further studies, involving a larger number of samples, are required to better understand whether these cells play a critical role in the immune response in canine seminomas. This is the first report to demonstrate the characteristics of T reg cell infiltration in canine seminoma.     

Molecular Characterization of Oxytocin Receptor Gene in Water Buffalo (Bubalus bubalis)

Buffaloes are known for their productivity as compared to average yielding cows due to higher fat percentage, better feed conversion ability and disease resistance. On the other hand, the reproductive performances of buffaloes are often considered as poor owing to late sexual maturity, weak/silent oestrus, repeat breeder and prolonged intercalving interval. The study of cascade of events during oestrus and oestrous cycle can be useful for the improvement of reproductive efficiency of buffaloes. More precisely, the hormonal changes initiated at the molecular level within the animal determine the reproductive nature of the species. Nucleotide/protein sequence analysis serves as a vital tool in analysing the binding of the hormones for their effect or functions. In this study, we have reported cloning and characterization of the complete coding (cDNA) sequence of oxytocin receptor gene (OXTR) in buffaloes. Buffalo OXTR gene contains an uninterrupted ORF of 1176 nucleotides corresponding to an inferred polypeptide length of 391 amino acids (aa). The molecular weight of the deduced aa sequence was found to be 43 kDa with an isoelectric point of 9.253 and 16.328 charge at pH 7.0. The deduced protein sequence consists of 38 strongly basic (+) (K,R), 22 strongly acidic (?) (D,E), 186 hydrophobic (A, I, L, F, W, V) and 95 Polar (N, C, Q, S, T, Y) aa. Results indicated that aspartate (D) at aa position 85 and D, R and C at aa positions 136, 137 and 138, respectively, are conserved in buffaloes. The buffalo OXTR gene shared a per cent similarity ranging from 84.7 to 98.1 and 88.5 to 97.7 at nucleotide and deduced aa sequence levels, respectively, with that of other species. Phylogram constructed on the basis of either nucleotide or deduced aa sequences of buffalo OXTR gene showed that buffalo, cattle and sheep have diverged from human and swine and formed a separate clad. The buffalo sequence has shown maximum similarity and closeness with cattle followed by sheep both at nucleotide and at aa level.     

IFN‐τ Acts in a Dose‐Dependent Manner on Prostaglandin Production by Buffalo Endometrial Stromal Cells Cultured in vitro

Interferon‐τ (IFN‐τ) has been recognized as the primary embryonic signal responsible for maternal recognition of pregnancy. Uterine endometrium produces both prostaglandin F_2α (PGF_2α) and prostaglandin E₂ (PGE₂). PGF_2α is responsible for the luteolysis; however, PGE₂ favours establishment of pregnancy by its luteoprotective action. In this study, the dose‐response effect of recombinant bovine IFN‐τ (rbIFN‐τ) on prostaglandin (PG) production by buffalo endometrial stromal cells cultured in vitro was studied. Buffalo endometrial stromal cells were isolated by double enzymatic digestion, initially with trypsin III followed by a cocktail of trypsin III, collagenase type II and DNase I and subsequently cultured till confluence. Further, cells were treated with different doses of rbIFN‐τ (0.001, 0.01, 0.1, 1.0 and 10 μg/ml) and keeping a separate set of control. Culture supernatant was collected after 6, 12 and 24 h of treatment. PG levels in the culture supernatant were measured by enzyme immune assay (EIA) and total cellular protein estimated by Bradford method. Results indicated that buffalo endometrial stromal cells following rbIFN‐τ treatment enhanced the secretion of both PGE₂ and PGF_2α, and also its ratio in a strict dose‐dependent manner with a significant increase (p < 0.01) in PGE₂ production at 1 μg/ml dose of rbIFN‐τ and maximal stimulation for both PG was observed at 10 μg/ml. Further, both PG production and its ratio were increased significantly (p < 0.01) in a time‐dependent fashion in all the groups at 6, 12 and 24 h post‐treatment with highest level achieved at 24 h as compared with control. Absolute levels of PGE₂ remained higher than PGF_2α indicating PGE₂ as the major PG produced by endometrial stromal cells. The dose‐dependent response of rbIFN‐τ signifies the importance of optimum concentration of IFN‐τ for the embryonic development especially during the critical period to establish successful pregnancy.     

Evaluation of genetic diversity among commercial cultivars, hybrids and local mango (Mangifera indica L.) genotypes of India using cumulative RAPD and ISSR markers

An assessment of genetic diversity studies was undertaken to understand the level and pattern of diversity in 65 mango (Mangifera indica L.) genotypes of India including 20 commercial cultivars, 18 hybrids, 25 local genotypes and two exotic cultivars based on qualitative and quantitative fruit characters as well as RAPD and ISSR profiles. A considerable variation was observed in respect of three important qualitative characters namely table quality, fruit attractiveness and storage life of ripe fruits and potentially superior genotypes for the above traits were identified. A wide variation was noticeable regarding metabolite composition of pulp of ripe mango fruit with respect to total soluble solids, total sugar, reducing sugar, acidity, sugar:acid ratio, ascorbic acid and phenolic content. Fifteen RAPD primers yielded 27 monomorphic and 129 polymorphic bands with percent polymorphism averaging 82.7%. Of a total 70 ISSR bands generated from eight ISSR primers, 60 bands (85.71%) were found to be polymorphic. Cumulative band data from these two methods precisely arranged accessions into eight clusters which correspond well with their pedigree relationship. UPGMA dendrograms drawn using RAPD, ISSR and cumulative data showed highly similar grouping of genotypes on the basis of their parental origin. No clear-cut geographical separation was revealed among East, West, North and South Indian mango cultivars by neither of these molecular markers nor their combinations. This supports the common gene pool origin of mango as well as operation of similar selection pressure as the cultivar preferences in these areas are largely similar.     

Activation of Zona‐Free Buffalo (Bubalus bubalis) Oocytes by Chemical or Electrical stimulation,and Subsequent Parthenogenetic Embryo Development

Parthenogenetic activation using zona‐free oocytes offers an alternative model that could be applied to develop protocols for the activation of reconstructed embryos for cloning. The aim of this study was to compare the efficacy of different methods for the activation of zona‐free buffalo oocytes in terms of their effects on the developmental competence of parthenogenetic embryos. The effects of zona removal on parthenogenetic activation and in vitro developmental competence of metaphase II oocytes were also examined. All activation methods were followed by incubation of 2 mm 6‐dimethylaminopurine (6‐DMAP) for 4 h. Out of three different pulse strengths (1.2, 2.1 or 3.3 kV/cm) used, 2.1 kV/cm resulted in the highest blastocyst rate (25.3%). On comparing different chemical agents and electric pulse, highest blastocyst rate was observed for calcium ionophore (CaI) (28.6%) followed by ethanol (25.0%), electric pulse (22.5%) and combined CaI and ethanol treatment (16.7%) although differences among them were not significant. Furthermore, a significantly reduced developmental potential was observed in zona‐free oocytes when compared to zona‐intact ones up to the blastocyst stage (44.3% vs 27.1%). In conclusion, zona‐free buffalo oocytes can be successfully activated for parthenogenetic development using chemical or electrical stimulation. Out of different agents examined, CaI followed by 6‐DMAP resulted in the highest blastocyst rate.     

Understanding Brown Planthopper Resistance in Rice: Genetics,Biochemical and Molecular Breeding Approaches

Brown planthopper (BPH, Nilaparvata lugens Stål) is the most devastating pest of rice in Asia and causes significant yield loss annually. Around 37 BPH resistance genes have been identified so far in indica, African rice varieties along with wild germplasms such as Oryza officinalis, O. minuta, O. nivara, O. punctata, O. rufipogon and O. latifolia. Genes/QTLs involved in BPH resistance, including Bph1, bph2/BPH26, Bph3, Bph6, bph7, BPH9, Bph12, Bph14, Bph15, Bph17, BPH18, bph19, Bph20, Bph21(t), Bph27, Bph27(t), Bph28(t), BPH29, QBph3, QBph4, QBph4.2, Bph30, Bph32, Bph33, Bph35 and Bph36, have been fine-mapped by different researchers across the globe. The majority of genes/QTLs are located on rice chromosomes 1, 3, 4, 6, 11 and 12. Rice plants respond to BPH attack by releasing various endogenous metabolites like proteinase inhibitors, callose, secondary metabolites (terpenes, alkaloids, flavonoid, etc.) and volatile compounds. Besides that, hormonal signal pathways mediating (antagonistic/synergistic) resistance responses in rice have been well studied. Marker-assisted breeding and genome editing techniques can also be adopted for improving resistance to novel BPH biotypes.     

中美不同年代大豆品种农艺性状的比较

选用地理纬度相似的中国辽宁省、美国俄亥俄州新近育成的大豆品种,分别和中国辽宁省20世纪20年代老品种进行了农艺性状的比较研究。结果表明:中美大豆品种主要农艺性状遗传改进的共有趋势是主茎荚数、单株荚数、3粒和4粒荚率、单株粒数、单株粒重、百粒重、粒茎比、收获指数明显增加。株高、主茎节数、节间长度和分枝数显著降低。美国新品种的单株荚数、单株粒数和粒茎比的增加幅度明显高于同期的中国新品种。而中国新品种的百粒重显著高于美国新品种。施肥水平对不同年代大豆品种主要农艺性状也有一定影响,且美国新品种在高肥条件下比中国新品种表现出更高的增产潜力。     

Accumulation of arsenic and its distribution in rice plant (Oryza sativa L.) in Gangetic West Bengal, India

The presence of arsenic in irrigation water and in paddy field soil were investigated to assess the accumulation of arsenic and its distribution in the various parts (root, straw, husk, and grain) of rice plant from an arsenic effected area of West Bengal. Results showed that the level of arsenic in irrigation water (0.05–0.70 mg l⁻¹) was much above the WHO recommended arsenic limit of 0.01 mg l⁻¹ for drinking water. The paddy soil gets contaminated from the irrigation water and thus enhancing the bioaccumulation of arsenic in rice plants. The total soil arsenic concentrations ranged from 1.34 to 14.09 mg kg⁻¹. Soil organic carbon showed positive correlation with arsenic accumulation in rice plant, while soil pH showed strong negative correlation. Higher accumulation of arsenic was noticed in the root (6.92 ± 0.241–28.63 ± 0.225 mg kg⁻¹) as compared to the straw (1.18 ± 0.002–2.13 ± 0.009 mg kg⁻¹), husk (0.40 ± 0.004–1.05 ± 0.006 mg kg⁻¹), and grain (0.16 ± 0.001–0.58 ± 0.003 mg kg⁻¹) parts of the rice plant. However, the accumulation of arsenic in the rice grain of all the studied samples was found to be between 0.16 ± 0.001 and 0.58 ± 0.003 mg kg⁻¹ dry weights of arsenic, which did not exceed the permissible limit in rice (1.0 mg kg⁻¹ according to WHO recommendation). Two rice plant varieties, one high yielding (Red Minikit) and another local (Megi) had been chosen for the study of arsenic translocation. Higher translocation of arsenic was seen in the high yielding variety (0.194–0.393) compared to that by the local rice variety (0.099–0.161). An appreciable high efficiency in translocation of arsenic from shoot to grain (0.099–0.393) was observed in both the rice varieties compared to the translocation from root to shoot (0.040–0.108).     

Ultrafiltration of Aqueous PVA Using Spinning Basket Membrane Module

The performance of spinning basket membrane (SBM) module was tested for the separation of polyvinyl alcohol (PVA) from wastewater. The SBM performance was examined using 50 kDa polyethersulfone ultrafiltration membrane under different parametric conditions. Also, the effects of rotational speed and transmembrane pressure on permeate flux and PVA rejection were investigated. The rotational speed played a significant role in decreasing membrane fouling by reducing the particle deposition on the membrane surface due to enhanced turbulence and shear force. Also, the in-built hydrodynamic cleaning facility of the SBM module allowed easy cleaning of the membrane. The steady-state value of percentage rejection of PVA was above 90% when the steady-state permeate flux value was above 54% of its initial value. The results suggested that spinning basket membrane module was efficient as well as economical for the separation of PVA from aqueous solution.     

Effect of testicular tissue lysate on developmental competence of porcine oocytes matured and fertilized in vitro

The objective of the present study was to investigate the effect of testicular tissue lysate (TTL) on developmental competence of germinal vesicle (GV) stage porcine oocytes. Two types of TTL were prepared through repeated freeze–thaw in liquid nitrogen, one from whole testicular tissue (wTTL) and other from either of four different sections of testes, namely just beneath the tunica albuginea (TA), from the transitional area between the seminiferous cord/tubules and the mediastinum testis (TR) and from the intermediate area (parenchymal tissue origin) and CE (cauda epididymis origin). The whole or section‐wise TTL treatments were given for 44 hr during in vitro maturation (IVM). Oocyte maturation was done in either of the two media, namely defined (high‐performance basic medium for porcine oocyte maturation, commercially available) and serum containing (TCM199). After maturation, oocytes were co‐incubated with fresh spermatozoa for 6 hr and then transferred to embryo culture media. Treatment of GV stage oocytes with wTTL (1 mg/ml) increased the cleavage and morula percentage rate (69.23 ± 6.23 and 48.15 ± 6.77, respectively) than that of their control (58.33 ± 8.08 and 32.54 ± 5.53, respectively) in defined media, and in serum‐containing media, cleavage and morula percentage rate were almost equal in both treatment (54.56 ± 7.79 and 34.70 ± 6.78, respectively) and control (59.52 ± 8.21 and 38.52 ± 6.54, respectively). However, effect of wTTL was not significant. In case of section‐wise TTL supplements, TR section significantly (p < .01) improved cleavage and morula rate (58.43 ± 7.98 and 36.14 ± 6.89, respectively) followed by TA. In conclusion, present study indicates that IVM, in vitro fertilization and in vitro culture of embryo are improved in the presence of TTL, particularly its TR section. Further study is expected to reveal the principal components of TTL which may prove useful for IVM.