Mohd Ashraf Rather

Chitosan-nanoconjugated hormone nanoparticles for sustained surge of gonadotropins and enhanced reproductive output in female fish.

A controlled release delivery system helps to overcome the problem of short life of the leutinizing hormone releasing hormone (LHRH) in blood and avoids use of multiple injections to enhance reproductive efficacy. Chitosan- and chitosan-gold nanoconjugates of salmon LHRH of desired size, dispersity and zeta potential were synthesized and evaluated at half the dose rate against full dose of bare LHRH for their reproductive efficacy in the female fish, Cyprinus carpio. Whereas injections of both the nanoconjugates induced controlled and sustained surge of the hormones with peak (P<0.01) at 24 hrs, surge due to bare LHRH reached its peak at 7 hrs and either remained at plateau or sharply declined thereafter. While the percentage of relative total eggs produced by fish were 130 and 67 per cent higher, that of fertilised eggs were 171 and 88 per cent higher on chitosan- and chitosan-gold nanoconjugates than bare LHRH. Chitosan nanoconjugates had a 13 per cent higher and chitosan gold preparation had a 9 per cent higher fertilization rate than bare LHRH. Histology of the ovaries also attested the pronounced effect of nanoparticles on reproductive output. This is the first report on use of chitosan-conjugated nanodelivery of gonadotropic hormone in fish.

Expression analysis of Sox9 genes during annual reproductive cycles in gonads and after nanodelivery of LHRH in Clarias batrachus

Transcription factor Sox9 plays a crucial role in determining the fate of several cell types and is a primary factor in regulation of gonadal development. Present study reports full-length cDNA sequence of Sox9a gene and partial coding sequence (cds) of Sox9b (two duplicate orthologs of Sox9 gene) from Clarias batrachus. The coding region of Sox9a gene encoded a peptide of 460 amino acids. The partial cds of Sox9b with the length of 558bp was amplified that codes for 186 amino acids. Quantitative Real-time PCR (qRT-PCR) analysis revealed that Sox9a and Sox9b mRNA expression was significantly higher in gonads and brain tissues. Furthermore Sox9a and Sox9b mRNA expression levels were high during preparatory and pre-spawning phases and decreased gradually with onset of spawning and post-spawning phases of reproductive cycles in gonads. Chitosan nanoconjugated sLHRH (CsLHRH) of particle size 133.0nm and zeta potential of 34.3mV were synthesized and evaluated against naked sLHRH (salmon luteinizing hormone-releasing hormone). The entrapment efficiency of CsLHRH was 63%. CsLHRH nanoparticles increased the expression level of Sox9 transcripts in gonads and steroid hormonal levels in blood of male and female. Thus, our findings clearly indicate that Sox9 genes play essential role during seasonal variation of gonads. Besides, the current study reports that sustained release delivery-system will be helpful for proper gonadal development of fish. To the best of our knowledge, till date no study has been reported on nanodelivery of sLHRH and their effect on reproductive gene expression in fish.

In silico analysis and expression studies of kisspeptin gene in C. catla

We report the characterization of kisspeptin gene which is considered to be essential for successful animal reproduction. The full-length cDNA sequence of kiss2 was 583 bp, consisted of 11 bp 5′-UTR (untranslated region) and 194 bp 3′-UTR, respectively. Open reading frame of 378 bp encoding a putative protein of 125 amino acids. The Catla catla kiss2 protein was having a molecular weight of 14.51 kDa and isoelectric point (pI) of 8.46. There were four serine (Ser), four threonine (Thr) and two tyrosine (Tyr) phosphorylation sites and no N-glycosylation sites on the predicted protein. The amino acids on positions 8, 11, 24, 80 and 114 were detected to be ligand binding sites. The signal peptide analysis predicted that C. catla kiss2 is a secretory protein. Kiss2 protein is localized in nuclear region (49.7%) and the extracellular region (38.3%) of the cell. Analysis of tissue distribution revealed that, kiss2 transcripts were predominantly expressed in the brain and gonads, with expression levels in female higher than those of male. Ontogenetic analysis of kiss2 demonstrated that expression level was low during early phase of development stages and more expression was observed during mature stage. Overall present results lay a strong basis for understanding the role of kisspeptin in the neuroendocrine system in teleosts.

RNA-Loaded Chitosan Nanoparticles for Enhanced Growth, Immunostimulation and Disease Resistance in Fish

Exogenous nucleotide supplementation during times of rapid growth and stress is preferred because de novo synthesis is insufficient and energetically a costly process. To overcome inefficient utilization of dietary nucleotides due to intestinal cell repulsion and dependency on pH, an efficient controlled delivery system based on chitosan nanoparticles (NPs) was developed. The effects of these (0.2% and 0.4%) RNA-loaded chitosan NPs (Chitosan: RNA ratio 2:1), 0.4% bare RNA, and 0.8% chitosan NPs on productive efficiency (growth rate, feed conversion and protein efficiency ratio), body composition, organo-somatic indices, haemato-biochemical and immune responses (WBC count, phagocytic activity, serum lysozyme, serum total protein and albumin: globulin ratio) and survival of Labeo rohita fish fingerlings, following challenge with pathogenic bacteria were evaluated. Dietary chitosan NPs were found not to affect productive efficiency, but improved (P 0.05) by dietary treatments. RNA-loaded chitosan NPs increased hepatoand viscerosomatic indices. The activity of metabolic enzymes (intestinal and liver alkaline phosphatase, alanineand aspartateamino transferases, lactateand malate-dehydrogeneases) corresponded with the performance of the respective diets. As growth, immunity and disease resistance in fish given dietary nano-sized RNA were significantly higher than in those given bare RNA or chitosan alone, and as nanoformulation reduced the usage of individual components by half, the use of RNA-loaded chitosan NPs can be favoured in the feed/food industry over chitosan and RNA alone.

Assessment of DNA Damage During Gene Delivery in Freshwater Prawn by Chitosan Reduced Gold Nanoparticles

Published by Oriental Scientific Publishing Company