Effects Of Supplemented Boron On The Development Of Spleen,Kidney,and Liver In The African Ostrich Chicks

1.Effects of boron supplementation on the spleenThe association between boron,spleen developmental changes,and the effects of boron(in the form of boric acid)on expression of Hsp40/70 was inspected in the present study.A total of thirty ostrich’s chick with similar body weight were arbitrarily allocated to six different groups,and fed boric acid(Omg,40mg,80mg,160mg,320mg,and 640mg/L,respectively).Histopathological analysis in the spleen was performed using HE technique.The expression amount of Hsp40/70 was checked by IHC and the technique of western blot,expression of the Hsp40/70 mRNA was studied by quantitative real-time PCR(qPCR)test.In order to study apoptosis;the labeling reaction of Dutp-biotin nickel labels(TUNEL)was analyzed in all treatment groups.The contents and results of this research work are following:1.1 Effects of boron on ostrich spleen tissue structureThere were not many differences in group Ⅱ compared to group Ⅰ.The white pulp number and volume was improved in group Ⅲ/Ⅳ as compared with the control group.The cells in marginal zone were dense and thickened,were separated with clear boundary.The histological alterations in the experimental group Ⅴ/Ⅵ were evident;the number and area of white pulp were condensed.The marginal zone boundary line was diminished.1.2 Effects of boron on distribution of Hsp40/70 in the ostrich chick spleenHsp70 located mainly in diffused form in the ostrich chick’s spleen and did not show many differences in the localization term between group Ⅰ and group Ⅱ,but the distribution arrangement of Hsp70 was evident in group Ⅲ,in particular group Ⅳ(160 mg/L)by comparing group Ⅰ.Conversely,the location of the positive signal for Hsp70 began to decrease in groups treated with high boron,chiefly in group Ⅵwith respect to group Ⅰ.Moreover,the positive products IOD revealed a slight enhancement in group Ⅱ which is non-significant.The mean density of Hsp70 signals were enhanced in group Ⅲ/Ⅳ,and were significantly higher.But,IOD in group V showed a non-significant decline,but a very significant decline was seen in group VI compared to control group.The tendency of Hsp70 level of expression by the technique of Western Blot was analogous to that of IHC staining.As the amount of boron increased,this expression of Hsp70 increased and reached a peak in group IV(160 mg).The expression trend of Hsp40 was similar to Hsp70;however as compared to Hsp70,the expression was less within the same groups.This shows that Hsp40 is co-factor of Hsp70 and aids in the activity of Hsp70.1.3 Effects of boron on mRNA expression of Hsp40/70 in ostrich spleenThe mRNA expression of Hsp40/70 in the group II were higher than control group.The expression in the group III were highly significant.The expression was at the highest level in group IV,and then began to decrease with least level was observed in group VI with respect to control group.This decrease in mRNA level presented that HSPs expression was dose-dependent,expression was initially induced at lower doses and then declined at high doses.1.4 Effects of boron on cell apoptosis in ostrich spleenTo verify the effects of boron on apoptosis in ostrich chicken spleen,the apoptosis was evaluated with the TUNEL method.The TUNEL cells were basically brown grains.The TUNEL signals were weaker up to 160 mg dose than control group,but were stronger in high dose groups.The IOD analysis of low doses,especially group IV showed a lower level,while the IOD level in groups V/VI was significantly higher than control group.2.Role of boron in the development of kidneyIn this study,the actions of boron on ostrich chicken kidney,especially in antioxidant capacity,were investigated.Thirty ostrich chickens were randomly divided into six equal groups and supplemented with the boric acid at the different concentrations in the drinking water.The relative oxidative/antioxidant enzymes(T-AOC,MDA,GSH-Px,CAT,GR,SOD)and cell apoptosis in kidney were determined.The expression of three important genes in this pathway(Nrf2,HO-1 and GCLC)were measured by qPCR,and the localization of key regulator Nrf2 was analyzed by IHC.The corresponding contents and results in the research are as follows:2.1 Effects of boron on apoptosis in ostrich chick kidney tissueThe apoptotic cells distributed in all places in kidney tissue which are basically granule cells of brown color.The no.of cell apoptosis were less in group Ⅱ and group Ⅲ boron groups as linked with control group.The number of apoptotic cells in 160 mg/L boron group increased slightly,whereas the number of apoptotic cells were considerably increased when the dose of boron was up to the 320 mg/L,and reached the peak at 640 mg/L dose.Furthermore,the IOD analysis of group Ⅱ and group Ⅲ showed decline in level with minimum level in group Ⅲ,while the level of mean density was highly significant in groups Ⅴ and Ⅵ,while associated to control group.2.2 Effects of boron on antioxidant activity in ostrich chick kidney tissueIn comparison to those of the control group,MDA content was significantly decreased by 26.02%and 48.12%in the 40 mg/L and 80 mg/L boron groups,respectively,and then increased at 160-320 mg/L,and doubled at 640 mg/L boron groups.The T-AOC activity in ostrich chick kidney tissue in 40 mg/L boron group was not different from that of control,and then significantly increased in 80 mg/L boron group.Conversely,the T-AOC activity were significantly declined in 640 mg/L boron group,which was opposite to that of the MDA content at the same group.The GSH-PX activity of ostrich chick kidney tissue were slightly increased in 40 and 80 mg/L boron groups,but not significantly different.However,GSH-PX in 160,320 boron groups increased significantly by～50%,and doubled in 640 mg/L group.2.3 Effects of boron on Nrf2 antioxidant pathway in ostrich chick kidney tissueThe results revealed that in low dose boron groups,Nrf2-positive products were mainly distributed in renal proximal tubules,while in high dose groups,the distribution of Nrf2 sandy particles was mostly in the epithelial cells.The IOD analysis revealed that localization of Nrf2 was high in low dose groups,while decrease in high dose groups as compared to group I.The qPCR analysis of three important genes in antioxidant pathway(Nrf2,HO-1,GCLc)revealed similar and intense difference among different groups.In comparison to the control group,addition of boron significantly increased the levels of Nrf2 in ostrich chick’s kidney tissue up to 160 mg/L dose,and peaked in 80 mg/L boron group.Aside from the 40 mg/L boron group,the levels of GCLc antioxidant in other boron groups all increased significantly and reached maximum in 80 mg/L boron group.Moreover,the effect of boron on HO-1 level was the largest in ostrich chick kidney tissue as compared to other antioxidant.3.Effects of boron on liver functioningThe African ostrich(Struthio camelus)were exposed to boric acid in drinking water(Omg,40mg,80mg,160mg,320mg and 640mg)to examine histological,apoptotic,histochemical,and serum biochemical parameters alterations induced by boron administration in the ostrich chick’s liver.Histological alteration in different groups were evaluated by HE and Periodic acid Schiff(PAS)technique of staining.The cellular apoptosis was detected by TUNEL assay reaction.The serum profile was assessed by spectrophotometer.The detailed RNA-Seq of data was done by transcriptomic method.The obtained findings are given below:3.1 Effects of boron on liver structural changesThe 90 d ostrich liver showed development in structure with abundant no.of cells and variable portal triads shapes.The development of liver was further seen in low dose boron groups with well-developed portal triad and hepatocyte area.The altered structure was seen in high dose groups.The major changes associated with boron intoxication includes;portal triads inflammation and alterations in hepatocytes.The portal triad’s inflammation was maximum in group VI.The mild anisonucleosis were seen in some hepatocytes of hepatic tissue,which was more in 640 mg/L dose group.Furthermore some hepatocytes also revealed nuclear vesiculation.Meanwhile,occasional nuclear pyknosis,binucleation,and necrosis were also observed.3.2 Effects of boron on glycogen contentsThe glycogen content in group Ⅱ was higher than group Ⅰ.The obvious differences were seen in group Ⅲ having maximum amount of glycogen content.The amount of glycogen started to reduce after group Ⅳ,which may be optimum dose.The group Ⅴ and group Ⅵ showed the least amount of glycogen.The results of glycogen levels were statistically confirmed by IOD analysis.3.3 Effects of boron on cellular apoptosis in the liver of ostrich chicksThe low dose groups showed less cellular apoptosis,with least cell death in group Ⅲ.The amount of apoptosis was more in high dose groups,as compared to control group.The statistical analysis by IOD support our TUNEL findings.Boron showed dose-dependent effect on apoptosis.The dose of boric acid beyond 160 mg elucidated more cellular apoptosis.3.4 Effects of boron on biochemical and transcriptomic parametersThe serum biochemical indices were significantly affected by boron supplementation.The results of enzyme activity parameter were obvious in high dose supplementation groups(320 mg,especially 640 mg/L boron).On the other hand,the parameters involved in metabolism showed favorable effects of boron in low doses group(80 mg).The RNA-Sequencing of 80 mg showed ameliorated effects of boron on ostrich chick.Overall results revealed positive effect of boron in low doses(80-160mg/L boric acid)for liver enzyme activity and metabolismIn short,overall results indicated that proper dietary boron intake(up to 160 mg)elucidated well developed histological structure,improvement in antioxidant capacity,less cellular apoptosis,and modulation in serum biochemical indices in the ostrich chicks.On the other hand,boron intoxication revealed obvious histological alterations.Furthermore,chronic boron showed decline in the HSPs expression,glycogen depletion,high apoptosis,and increase in enzyme activity;which may be associated with boron stress.