Teleost Nonapeptides, Isotocin and Vasotocin Administration Released the Milt by Abdominal Massage in Male Catfish, Clarias magur.

In the present work the nonapeptides i.e., isotocin and vasotocin alone or in a combination were tested in C. magur to evaluate their effect on stripping by abdominal massage. Also, we used chitosan-carbon nanotube nanocomposites to conjugate the nonapetides isotocin (abbreviated as COOH-SWCNTCSPeP) and isotocin and vasotocin (COOH-SWCNTCSPePs) with the aim of sustaining the effect for a longer duration. The conjugation of nonapeptides with nanocomposites was confirmed by Fourier-transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), Thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS). Two experiments were conducted to study the effect of naked (without nanoparticles) and conjugated nonapeptides on the milt release by stripping. Both the experiments consisted of eight treatments which included four naked groups two nanoconjugated groups and two controls. Both naked and nonconjugated formulations were successful in stripping the male catfish. The mRNA expression of selected reproductive genes was analysed to decipher the effect of nanopeptides at the molecular level. Nonapeptide treatment either naked or nanoconjugated, resulted in the upregulation of the transcript level of genes. Histological analysis revealed the concentration of spermatozoa was more in peptide injected groups than in the controls. The synergistic effects of nonapeptides and Ovatide had a positive impact on GSI. Thus, the present formulations were successful in stripping the male catfish to obtain the milt with significant reproductive success. Even though the naked groups perform better but the number of males required to fertilize the eggs in nanoconjuagted groups was smaller making it worth using for the delivery of nonapeptides.

Chitosan-eurycomanone nanoformulation acts on steroidogenesis pathway genes to increase the reproduction rate in fish.

The study was undertaken to explore the molecular mechanism of eurycomanone, a major compound of Eurycoma longifolia plant in increasing the reproductive processes in the male fish model. Chitosan-nanoconjugated eurycomanone nanoparticles with a significant particle size [130 nm (CED1); 144.1 nm (CED2)] and stable zeta potentials (+49.1 mV and +30 mV) were synthesized and evaluated against naked eurycomanone (ED1 and ED2). In present study, short-term and long-term experiments were conducted to evaluate the effect of nano-formulation on expression of endocrine-related genes, circulating hormone concentrations (Follicle stimulating hormone, FSH; luteinizing hormone, LH; progesterone, testosterone and 17-ß estradiol) and reproductive capacity of male Clarias magur. In short-term experiment, the sampling of tissues was done on hourly basis after injection of eurycomanone either alone or with chitosan and long-term experiment was carried for 21 days and in this the injection was repeated after 7 days and 14 days. Treatments CED1 and CED2 showed controlled and sustained surge of the transcript level of selected genes (except aromatase) and serum hormones (except 17ß-estradiol) compared to ED1 and ED2 groups. The transcript levels of aromatase and serum 17ß-estradiol hormone showed the declining trend in the chitosan conjugated groups. The gonadosomatic index (GSI), reproductive capacity, intracellular calcium and selenium and cellular structure of testes were improved in CED1 and CED2 groups compared to other treatments. Furthermore, the effect of chitosan conjugated eurycomanone was evaluated in primary testicular cells and an increase in the mRNA expression level of endocrine-related genes was detected. This is the first report of the use of chitosan conjugated eurycomanone and present study elucidates the molecular mechanism of eurycomanone in increasing the reproductive output in animals.

Fabrication and characterization of chitosan conjugated eurycomanone nanoparticles: In vivo evaluation of the biodistribution and toxicity in fish.

Chitosan nanoparticles (CNPs) have been proven considerable delivery agents due to their remarkable physicochemical properties. Present study reports the fabrication of CNPs by ionic gelation process and their characterization by different approaches. The constructed nanoparticles were successfully conjugated with eurycomanone with significant entrapment efficiency. Particle size of chitosan and chitosan conjugated eurycomanone nanoparticles were 126.2nm and 130nm respectively. Scanning electron microscopy showed that the particles were spherical in shape and well dispersed. Cross-linking between CNPs and eurycomanone (CENPs) were confirmed by Fourier-transform infrared (FTIR) spectroscopy. Fluorescent nanoparticles were prepared by using Rhodamine-6G dye, characterised by SEM and confirmed for conjugation by FTIR. Biodistribution of CENPs showed the presence of fluorescent nanoparticles in liver, kidney, testes and brain of C. magur. The toxicity of CENPs was evaluated by comparing the histological sections of catfish testes collected from treated and control group. No signs of toxicity were seen in testes after the delivery of CENPs. Molecular docking study revealed high spontaneous binding ability of chitosan with eurycomanone and aromatase enzyme. The study reports that CNPs can act as a stabilizing agent for eurycomanone formulation and could be a promising approach to increase the reproductive performance of the fishes.

Cloning, characterisation, docking and expression analysis of 3-beta-hydroxysteroid dehydrogenase during ontogenetic development and annual reproductive cycles in catfish, Clarias batrachus.

Fish like higher animals, have a well-defined mechanism to produce sex steroids that play a critical role in gonadal development and maturation. In this study, we aimed to analyse the expression pattern of 3ß-HSD in different tissues, during ontogenetic development and gonadal recrudescence of Clarias batrachus. A full-length cDNA of 1617 bp including an open reading frame (ORF) of 1125 bp encoding 374 amino acids was isolated from testes of C. batrachus. The docking analysis between C. batrachus 3ß-HSD protein and eurycomanone exhibited high binding affinity toward each other with total energy of -108.292 kcal/mol and van der Waals (VDW) interaction of -84.2838 kcal/mol. The 3ß-HSD transcript level during ontogeny was detected in all the stages starting from the fertilized egg. The mature C. batrachus showed more expression of 3ß-HSD mRNA in gonads and brain while weak expression was detected in the remaining tissues analysed. The 3ß-HSD mRNA expression during annual reproductive phases of gonads was more in preparatory and pre-spawning stages than that of spawning and post-spawning phases. The mRNA expression results together suggest that 3ß-HSD plays an important role in gonadal development. Furthermore, the active binding sites on 3ß-HSD protein could be targeted in pharmacological drug designing to cope with reproductive dysfunctions in fish.