Development and validation of a sensitive enzyme immunoassay (EIA) for blood plasma cortisol in female cattle,buffaloes, and goats

A highly sensitive enzyme immunoassay (EIA) that used the second antibody coating technique and the cortisol-horseradish peroxidase conjugate as a label for determination of free and total cortisol in blood plasma of dairy animals (cows, buffaloes, and goats) was developed. For biological validation of the EIA, blood samples were collected from the animals at 48 and 24 h before and 0, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, and 132 h after dexamethasone administration. The EIA was performed directly with 20 μL of fresh plasma (for free cortisol) and also with 20 μL of heat-treated plasma (for total cortisol) after 1:5 dilutions with PBS. Cortisol standards ranging from 0.39 to 200 pg/well/20 μL were used, and the sensitivity of the EIA procedure was found to be 0.39 pg/well/20 μL, which corresponded to 0.02 ng/mL. In comparison with RIA the EIA was at least 4 times more sensitive and required 5 times less cortisol antiserum. In female cattle, buffaloes, and goats, the total, free, and bound plasma cortisol before dexamethasone administration was significantly (P < 0.05) higher than the total, free, and bound cortisol after dexamethasone administration. It can be concluded from these studies that the direct, sensitive EIA validated for estimating the free and total cortisol concentrations was sufficiently reliable and quick for studying the dynamics of cortisol distribution in blood plasma of dairy animals.     

Aquaculture and stress management: a review of probiotic intervention

To meet the ever‐increasing demand for animal protein, aquaculture continuously requires new techniques to increase the production yield. However, with every step towards intensification of aquaculture practices, there is an increase in stress level on the animal as well as on the environment. Feeding practices in aqua farming usually plays an important role, and the addition of various additives to a balanced feed formula to achieve better growth is a common practice among the fish and shrimp culturists. Probiotics, also known as ‘bio‐friendly agents’, such as LAB (Lactobacillus), yeasts and Bacillus sp., can be introduced into the culture environment to control and compete with pathogenic bacteria as well as to promote the growth of the cultured organisms. In addition, probiotics are non‐pathogenic and non‐toxic micro‐organisms, having no undesirable side effects when administered to aquatic organisms. Probiotics are also known to play an important role in developing innate immunity among the fishes, and hence help them to fight against any pathogenic bacterias as well as against environmental stressors. The present review is a brief but informative compilation of the different essential and desirable traits of probiotics, their mode of action and their useful effects on fishes. The review also highlights the role of probiotics in helping the fishes to combat against the different physical, chemical and biological stress.     

Influences of incorporating detoxified Jatropha curcas kernel meal in common carp (Cyprinus carpio L.) diet on the expression of growth hormone‐ and insulin‐like growth factor‐1‐encoding genes

Jatropha curcas is a drought‐resistant shrub or small tree widespread all over the tropics and subtropics. The use of J. curcas (L) kernel meal in fish feed is limited owing to the presence of toxic and antinutritional constituents. In this study, it was detoxified using heat treatment and organic solvent extraction method. The detoxification process was carried out for 60 min to obtain the detoxified meal. Cyprinus carpio L. fingerlings (n = 180; avg. wt. 3.2 ± 0.07 g) were randomly distributed in five treatment groups with four replicates and fed isonitrogenous diets (crude protein 38%) for 8 weeks. The inclusion levels of the detoxified Jatropha kernel meal (DJKM) and soybean meal (SBM) were as follows: control diet was prepared with fish meal (FM) and wheat meal, without any DJKM and SBM; diets S₅₀ and J₅₀: 50% of FM protein replaced by SBM and DJKM respectively; diets S₇₅ and J₇₅: 75% of FM protein replaced by SBM and DJKM respectively. Highest body mass gain and insulin‐like growth factor‐1 (IGF‐1) gene expression in brain, liver and muscle were observed for the control group, which were statistically similar to those for J₅₀ group and significantly (p < 0.05) higher than for all other groups, whereas growth hormone gene expression in brain, liver and muscle exhibited opposite trend. Insulin‐like growth factor‐1 concentration in plasma did not differ significantly among the five groups. Conclusively, growth performance was in parallel with IGF‐1 gene expression and exhibited negative trend with GH gene expression.     

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.     

Antibacterial and Cytotoxic New Napyradiomycins from the Marine-Derived Streptomyces sp. SCSIO 10428

Three new napyradiomycins (1–3) were isolated from the culture broth of a marine-derived actinomycete strain SCSIO 10428, together with six known related analogues napyradiomycin A1 (4), 18-oxonapyradiomycin A1 (5), napyradiomycin B1 (6), napyradiomycin B3 (7), naphthomevalin (8), and napyradiomycin SR (9). The strain SCSIO 10428 was identified as a Streptomyces species by the sequence analysis of its 16S rRNA gene. The structures of new compounds 1–3, designated 4-dehydro-4a-dechloronapyradiomycin A1 (1), 3-dechloro-3-bromonapyradiomycin A1 (2), and 3-chloro-6,8-dihydroxy-8-α-lapachone (3), respectively, were elucidated by comparing their 1D and 2D NMR spectroscopic data with known congeners. None of the napyradiomycins 1–9 showed antioxidative activities. Napyradiomycins 1–8 displayed antibacterial activities against three Gram-positive bacteria Staphylococcus and Bacillus strains with MIC values ranging from 0.25 to 32 μg mL⁻¹, with the exception that compound 3 had a MIC value of above 128 μg mL⁻¹ against Staphylococcus aureus ATCC 29213. Napyradiomycins 2, 4, 6, and 7 exhibited moderate cytotoxicities against four human cancer cell lines SF-268, MCF-7, NCI-H460, and HepG-2 with IC₅₀ values below 20 μM, while the IC₅₀ values for other five napyradiomycins 1, 3, 5, 8 and 9 were above 20 μM.     

Screening of genotypes of menthol mint Mentha arvensis for high yields of herbage and essential oil under late cropping conditions of the sub-tropical Indo-Gangetic plains

Summary

The essential oil yields and expression of related characters were compared for seven cultivar genotypes of menthol mint Mentha arvensis using two methods of planting in the winter rabi – summer season (October to July) in a sub-tropical agroclimatic environment. The crops of all the cultivars were planted in the field by (1) sowing of suckers on 2 January and (2) transplanting germinated pieces of sucker at different times between 17 March to 14 April. Staggering of transplanting time up to 7 April did not affect oil yields and the related plant growth properties of mint crops. The oil yields of the crops planted on 14 April were lower by about 30%. In the early sucker planted crops, the oil yields were about 30% higher than those obtained from the transplanted crops of 17 March to 7 April and about double that obtained from crops transplanted on 14 April. The oil yields from the crops of the superior genotype Kosi were equal to or higher than the corresponding means of all genotypes under both planting methods. The oil yield from the crops of Kosi genotype obtained by sucker sowing method was estimated as 333 kg ha^–1. The corresponding average yield from the crops of this genotype obtained by transplanting of germinated suckers between 17 March and 7 April was about 293 kg ha^–1 and that from the crop transplanted on 14 April was 218 kg ha^–1. With the Kosi genotype, the latter two types of transplanted mint crops gave oil yields lower only by about 12% and 33% compared with the long-duration sucker-sown crop. It is concluded that a crop of mustard (brassica), Bengalgram (chickpea) or wheat sown between October and November and harvested between early March to middle of April could be taken before cropping of the Kosi genotype of M. arvensis by plantlet transplanting. These results demonstrate the potential of the following rotation of crops in the sub-tropical environments: from June/July/August to October/November (kharif cropping season), rice, maize, sorghum or pigeonpea; from November/December to February/March, mustard and Bengalgram or from November/December to April (rabi cropping season), wheat; from March/April to June/July (zaid cropping season), transplanted menthol mint.     

Comparison of seed-derived with micropropagated male-sterile plants of Tagetes erecta L. for F1 hybrid seed production

Summary

To multiply large number of male-sterile marigold plants for F₁ hybrid seed production, an efficient protocol for in vitro cloning of field-grown differentiated male sterile plants has been developed. A comparative field performance study of tissue culture and seed-derived male sterile plants of two marigold genotypes was undertaken to test the possibility of using micropropagated plants in hybrid seed production. Tissue culture raised plants of both genotypes had superior field performance to the seed-derived counterparts. These plants were more vigorous in growth, i.e. in terms of plant height, number of secondary branches and number of leaves and plant spread, while the leaf chlorophyll contents were equal to that of seedling plants. Flowering was earlier by 2-3 weeks and the number of flowers per plant was also higher in such plants. Repeated hand pollination of sterile flowers with bagged flowers of cv. Pusa Narangi Gainda showed that seed set and bold seed yield were higher or almost comparable with the seed-derived plants. The results clearly indicate that the tissue culture can be adopted for the successful cloning of male-sterile plants, which could then be utilized for producing F₁ seeds with higher quantities of bold seeds with better storability.     

Optimising parameters for biolistic gun-mediated genetic transformation of tea [Camellia sinensis (L.) O. Kuntze]

Summary

Genetic improvement of tea through breeding is difficult. Therefore, transgenic tea plants expressing the osmotin gene from Nicotiana tabacum were produced using parameters optimised for biolistic-gun mediated transformation. During optimisation, a total of 4,500 somatic embryos were bombarded using nine combinations of variable target distances and burst pressures, while keeping the gap distance (0.6 cm) and macrocarrier flight distance (16 mm) constant.A total of 90 independent, PCR-positive lines were generated. Southern hybridisation confirmed integration of the osmotin gene in 26 out of 27 PCR-positive lines (three independent lines from each of the nine parameter combinations were selected at random). Statistical analysis revealed that the efficiency of transgene integration was significantly affected by target distance. Only those lines derived from somatic embryos bombarded with 1.0 µg plasmid DNA using a 7.58 MPa burst pressure and 9-cm target distance showed osmotin expression. This was evident from strong northern hybridisation and RT-PCR signals. Leaves of 4-year-old transgenic plants growing in a contained polythene tunnel showed improved osmotic adjustment in response to osmotic stress imposed by NaCl. The osmotic potentials of transgenic leaves immersed in 100 mM or 200 mM NaCl solutions were more negative than those of non-transformed control leaves.