Biological and toxicological effects of dietary organic and inorganic forms of cobalt on Tor putitora

Abstract

Minerals play important biological and physicochemical functions inside living organisms. However, their efficiency depends on many factors, including chemical form, particle size, and dosage level. Cobalt is one of the important essential trace minerals, required in low quantity in the diet for normal body functioning. Unfortunately, information regarding the optimal dietary supplementation level of cobalt in most fish species, including Mahseer Tor putitora, is not documented. Here an attempt has been made to determine the optimum dietary supplementation level of different chemical forms of cobalt in Tor putitora. First, a feeding trial was executed to determine the optimum dietary dosage level of the conventional inorganic form of cobalt (macro scale). Advanced fry of T. Putitora were fed graded levels of cobalt chloride supplemented diets (0.5-3mg/kg diet) for 90 days. Results indicated the positive effect of cobalt supplemented diet at dosage level up to 2mg/kg feed on the growth performance and proximate body composition of fish. However, Co supplemental level above 2mg/kg reduced the growth rate of fish, indicating a dose dependent toxic effect. Moreover, a curvilinear relationship was observed with the increasing cobalt supplementation in diets between whole body crude protein (%) content and weight gain (%) of fish. Furthermore, at higher supplementation levels, results indicated the inhibitory effect of cobalt on the accumulation of iron and manganese in the muscle of fish. To validate the results, a nutrigenomic study was designed and the effect of dietary cobalt supplementation on the expression of growth-related genes, i.e., myoblast determination protein 1 homolog MyoD and myogenin MyoG in the muscle and stress response gene (heat shock protein 70KDa) in the liver was also studied. Results indicated the positive effect of a 2mg Co/kg diet on the mRNA levels of growth genes in the muscle. However, cobalt supplementation at a higher dosage level (3mg/kg diet), down-regulated the growth-related genes in the muscle and up-regulated the expression of stress response gene (heat shock protein 70KDa) in the liver of fish. Results of our preliminary experiment revealed that CoCl2 supplementation at 2 mg/kg diet is optimal for the muscle growth xxi and body composition of T.putitora. We then further extended our investigation by studying the effect of graded levels of different chemical forms of cobalt, i.e., cobalt chloride nanoparticles (Co NPs) and cobalt methionine complex (Co-Met) on T. putitora fingerlings. First, Co-NPs and Co Met complex was prepared by using ball-milling and precipitation methods respectively, and then characterized by using X-rays diffraction spectroscopy (XRD), scanning electron microscope (SEM), and energy dispersive spectroscopy (EDS) for their size, surface morphology, and elemental composition. Characterization of particles indicated the crystalline nature of Co-NPs with particle size <20nm. However, the XRD spectrum of the Co-Met complex did not show any peak, indicating the amorphous nature of the complex. SEM image of Co-Met complex appeared as a honeycomb-like octahedral structure with an average size of 82.69nm. Furthermore, EDS spectrums indicated 21% oxygen, 40.33 % chloride and 37% cobalt in Co-NPs, and 31% carbon, 1.32% nitrogen, 48.53 % oxygen, 2.06% cobalt and 0.31% chlorine in Co-Met complex. In the next phase of the study, feeding trials were conducted to study the effect of graded levels of Co-NPs and Co Met complex supplemented diets on T. putitora fingerlings. Results indicated the significant effect (P<0.05) of both chemical forms of cobalt on the growth performance, hemato-immunological indices, intestinal enzyme activities, the proximate composition of muscle, and metals accumulation in muscle. Co-NPs supplemented diet showed a positive effect on fish up to the inclusion level of 1.5mg/kg diet. However, further inclusion of Co-NPs in diet-induced toxicological effects, i.e., negative effect on growth, hemato-immunological indices, intestinal enzyme activities, and proximate composition of the muscle. Contrary to that, the Co-Met complex supplemented diet did not show any toxicity even at the maximum dosage level, i.e., 3mg /kg diet. Moreover, after exposure to a pathogen, Aeromonas hydrophila, maximum mortality was observed in a group of fish reared on 3mgCo-NPs /kg diet. However, the Co-Met fed-group of fish reared on same dosage level (3mgCo-Met complex /kg diet), showed no mortality. Two way ANOVA indicated a significant effect of chemical forms and dosage levels on all studied parameters of fish. Moreover, a xxii significant (P<0.05) interaction between both variables (chemical forms × dosage levels) indicated how both of them affected the fish. Furthermore, a pairwise comparison revealed that the organic form of cobalt (Co-Met) at every dosage level showed a comparatively stronger positive effect than the inorganic nanoform of cobalt. For further validation of results and practical application of both chemical forms of cobalt supplemented diets, a further completely randomize feeding experiment was designed and conducted in earthen ponds in semi-intensive culture conditions. Based on previous experiment results, two dosage levels of both organic and inorganic forms of cobalt, i.e., 1.5mg and 3mg /kg diet were selected and fed to fish for 90 days. Results indicated a dosage dependent positive effect of Co-Met complex supplemented diets on growth performance (Wf, %WG, AWG, ADG, %SGR), hematological indices (RBC, HB, HCT, MCV, MCH, MCHC immunological indices (plasma protein, IgM level, serum lysozyme activity, WBCs count, respiratory burst and phagocytic activity), liver function indices (serum AST and ALT activities), nutritive value (muscle proximate composition and amino acids profile) and expression of MyoD and MyoG genes in the muscle of fish. However, in accord with the previous results, Co-NPs supplemented diet showed a positive effect up to inclusion level of 1.5mg/kg diet, and at higher inclusion level (3mg/kg diet), showed a toxic effect on all studied parameters of fish. Overall, Co NPs appeared more efficient and required in less quantity for the beneficial and toxicological effects than conventional CoCl2. However, Co-Met complex as compared to Co-NPs showed the most pronounced positive effects without any toxicity at all dosage levels on all studied parameters of fish. Based on the results, we could recommend an organic form of Co (Co-Met complex) as a dietary supplement for improving growth, immunity, health status, and nutritive value of T.putitora