Diagnosis Of Selenium Deficiency Disease And Assessment Of Multi-Organ Damage In Calves On A Cattle Farm

Selenium performs important biological functions in maintaining body health.Calves are more susceptible to selenium deficiency because of their rapid growth rate compared with adult cows,which have a more acute disease course and severe clinical symptoms,often resulting in massive mortality within a short period of time and causing huge economic losses.There are few studies on the damage mechanism of selenium deficiency disease in calves and even dairy cows,therefore,this study was conducted to investigate the damage mechanism of heart,liver,lung and intestine of calves under the condition of selenium deficiency without secondary infection.In this study,selenium levels in food,blood,and organs of calves were measured by ICP-MS,and the results demonstrated that selenium deficiency in calves was closely linked to a severe lack of selenium in feeds and alfalfa.The main clinical symptoms of selenium-deficient calves were depression,loss of appetite,diarrhea,respiratory distress,edema of the limbs,lying down or refusing to exercise,and the heart sounds were subtle and disorganized.The disease had an acute onset and cause sudden death in severe individuals.Autopsy results showed multiple organs were damaged in the affected calves:massive pleural effusion,pulmonary congestion;pericardial effusion,ventricular dilatation,pale and hard myocardium with necrosis;liver congestion,swelling and hardness;intestinal hemorrhage with cicatricial lesions.Histological analysis showed that the myocardium of selenium-deficient calves had extensive necrosis and disappearance of normal structures;the arrangement of liver lobules was disturbed,hepatic sinusoids were congested,hepatocytes were swollen and necrotic,and some hepatocyte nuclei were dissolved and disappeared;alveolar epithelial cells were swollen,capillaries were congested,alveolar septum was thickened,and diffuse interstitial inflammation appeared;the structure of small intestinal villi was broken,hemorrhagic,epithelial cells were detached,necrotic,and goblet cells almost disappeared.All the affected organs showed different degrees of inflammatory cell infiltration.In this study,selenoprotein gene and partial selenoprotein expression levels were detected using RT-PCR and WB.The results showed that the changes of selenoprotein expression in seleniumdeficient calves were tissue-specific,but most of the down-regulated selenoproteins were associated with redox reactions.To further investigate the redox status of calves with selenium deficiency,this study examined the changes of some redox-related indicators,including the activities of catalase,superoxide dismutase,and thioredoxin reductase,total antioxidant capacity,and the concentrations of reduced glutathione,hydrogen peroxide,and malondialdehyde,using commercial kits.The results showed that the levels of antioxidant enzymes and total antioxidant capacity were significantly reduced and the concentrations of by-products of oxidative stress(hydrogen peroxide and malondialdehyde)were significantly increased in the organs of selenium-deficient calves.In addition,the m RNA and protein levels of oxidative stress-related proteins COX-2,HIF-1α and i NOS were significantly increased.All of these results suggested that selenium-deficient calves were in a severe state of oxidative stress.Inflammation and oxidative stress are inextricably linked conditions.Therefore,this study used ELISA kits and RT-PCR to detect the expression changes of inflammation-related indicators under selenium deficiency conditions.The results showed that the pro-inflammatory cytokines IL-1β,IL-6,IL-8,IL-12,and TNF-α were significantly increased,while the anti-inflammatory cytokines TGF-β and IL-10 were significantly reduced.In addition,this study also examined the changes of two important inflammation-related pathways,namely NF-κB pathway and MAPK pathway,under selenium deficiency conditions.The results showed that both pathways were activated to different degrees in selenium-deficient calves,while P50,P65,and other NF-κB-related proteins and MAPK pathway-related proteins JNK and P38 were up-regulated.In this study,the cell death modes in various organs of selenium-deficient calves were investigated.TUNEL staining showed that severe apoptosis occurred in all examined organs of selenium-deficient calves,with the heart being the most affected and the lung being the least.The expression changes of genes and proteins related to the apoptosis pathway were detected by RTPCR and WB,and the results showed that the Caspase-8 and Caspase-9 apoptosis pathways were activated in various organs of selenium-deficient calves,with the expression of Bax,Bak,Caspase-3,Caspase-7,Caspase-8,Caspase-9,and APAF1 being up-regulated,while anti-apoptotic proteins XIAP and Bcl-2 were down-regulated.In addition,necroptosis occurred in all organs of seleniumdeficient calves,and the expression of genes or proteins related to this death mode,such as MLKL,RIPK1,and RIPK3,were significantly up-regulated.In the present study,an in-depth study of calves revealed that calves organs under selenium deficiency conditions have similar pathogenesis.Selenium deficiency resulted in affected expression of selenoproteins in different organs of calves,and most of the down-regulated selenoproteins were associated with oxidative stress,with severe oxidative stress occurring in the heart,liver,lungs,and intestine of selenium-deficient calves.Under oxidative stress conditions,tissues and cells of selenium-deficient calves were damaged,pro-inflammatory cytokines were upregulated and anti-inflammatory factors were down-regulated,while the NF-κB pathway and the MAPK pathway were activated.In addition,selenium deficiency caused regulated cell death in calves,with activation of Caspase-8 and Caspase-9 apoptotic pathways,accompanied by necroptosis.Oxidative stress,inflammation,apoptosis and necroptosis ultimately lead to tissue lesions.