Biochemical parameters
The alterations in total serum protein, albumin, globulin and A:G ratio in different groups are presented in Table 1.
There was a significant reduction in total protein as well as albumin while globulin levels remained unaffected levels in groups fed ionophores. Vitamin C significantly improved total protein in both ionophore groups but albumin levels were significantly better only in salinomycin group. Others have also reported that concentration of total protein, albumin and globulin were decreased because of salinomycin toxicity
(Arun et al., 2003). The data on alterations in biochemical parameters indicative of liver and kidney damage is presented in Table 2. Creatinine, ALP, AST and ALT, levels were found to be significantly raised in birds fed maduramicin and salinomycin at 2
nd WPE. Vitamin C addition significantly reduced levels of all variables except creatinine.
Oxidative stress parameters
The average data of SOD, CAT, GPx and MDA in blood of birds of different groups is summarized in Table 3. The average data of SOD, CAT, GPx and LPO in the liver of birds in different groups as observed at 2
nd WPE is summarized in Table 4. There was a significant increase in values of SOD, CAT and LPO in groups fed maduramycin and sainomycin whereas significant improvement was seen with concomitant vitamin C feeding in CAT and SOD values. No significant changes were registered in GPx levels after maduramycin toxicity when compared to control birds however salinomycin caused a significant increase in GPx as compared to control. Vitamin C completely ameliorated raised GPx concentrations in salinomycin fed birds. Average SOD, CAT, GPx and LPO levels in the kidneys of birds at 2
nd WPE is summarized in Table 5. Group III and V had significantly increased SOD, CAT, GPx and LPO levels as compared to control group but vitamin C caused significant amelioration of SOD, CAT, GPx and LPO levels. Free radical mediated cell damage is an important player in the pathogenesis of various disease conditions
(Sood et al., 2019). Oxidative damage is kept in check by antioxidant machinery which consists of enzymes like SOD, CAT and GPx which scavenge reactive oxygen species (ROS) such as O
2-, H
2O
2 etc generated in response to xenobiotic metabolism (
Yang et al. 2024). There was a significant increase in SOD, CAT, GPx and LPO values after giving maduramycin. Similar alterations were recorded in salinomycin group except for CAT levels which remained unaffected. Vitamin C supplementation partially but significantly ameliorated both the ionophore induced alterations. However, in a study by
Ni et al. (2019), significantly increased activities of SOD, CAT, GPx and GST in the liver of zebrafish were observed after maduramicin toxicity and
Kamashi et al. (2004) also reported increased levels of GPx, GSH and CAT in blood in chicks receiving toxic doses of salinomycin, which are in concurrence with our results. It has been shown that addition of ginseng, can ameliorate the oxidative stress caused by maduramicin
(Sivakumar et al., 2007) which is similar to our results where vitamin C significantly reduced oxidative stress caused by maduramicin and salinomycin. Many previous studies have also shown that dietary supplementation with antioxidants mitigates oxidative stress and improves broiler production
(Gopi et al., 2019; Choudhary and Singh, 2017).
Immune response parameters
The HI test showed robust titer specific to NDV ranging from 2
6 to 2
7 in all the vaccinated groups. There was a decrease in HI titer in group III and V (2
6) as compared to group I and group II (2
7) whereas group IV and VI showed same titer as that of control group birds. The data of skin thickness measurements in various groups is presented in Table 6 and Fig 1a and 1b. A significant reduction in skin thickness in response to PHA-P inoculation in the interdigital space was seen in ionophore only administered groups. Vitamin C co-administration with either ionophore improved the humoral immune response. These results were also supported by severe lymphocyte depletion in spleen and bursa after ionophore administration and lack of such changes in birds co-treated with vitamin C. Similar findings have also been reported by
Koutoulis et al. (2013) who saw splenic atrophy in broiler breeders and turkeys because of salinomycin toxicity.
Rahman and Joshi (2010) also showed that lead acetate suppressed both humoral response and cell mediated response in broiler birds.
Pathomorphological observations
A comparison of the lesion scores (heart, skeletal muscles, gizzard, kidney, liver and spleen) in different groups is presented in Table 7.
Heart
In group III and V, severe hemorrhages on epicardium were observed and multifocally, subepicardium had aggregates of heterophils and lymphocytes and myocardial interstitium was expanded by congestion, hemorrhages and perivascular edema. Myocardial fibres showed multifocal extensive segmental degeneration and coagulation necrosis. Cardiac myofibers had homogeneously hyalinized sarcoplasm with pyknotic or faded nuclei which were often fragmented with loss of cross striations and infiltrated by predominantly heterophils and fewer macrophages (Fig 2a, 2c). In groups IV and VI, the pathological changes were subdued as only mild congestion and degeneration was seen and focal necrotic areas were occasionally found (Fig 2b, 2d).
Agaoglu et al. (2002) also found petechiae in the heart and focal muscle necrosis in goat heart after accidental intake of salinomycin in feed. Post mortem examination of heart in camels revealed pale and flabby heart and presence of congestive heart failure after accidental salinomycin toxicity (
Al-Wabel 2012). Similarly,
Chen et al. (2014) also observed cardiomyocyte damage and heart failure after maduramicin toxicity and
Shimshoni et al. (2014) also reported degenerative changes in the myocardium of pregnant gilts after toxicosis of maduramicin. Such type of changes in the heart may be due to ionic (Na
+, K
+ or Ca
+) imbalance caused by toxicity of ionophores since heart is more prone to ionic disturbances (
Al-Wabel 2012). In heart of sheep, there was extensive subepicardial and myocardial hemorrhages, cardiomyolysis and myocarditis was seen by
Ashrafihelan et al. (2014) due to accidental intake of salinomycin. Hemorrhages in heart were seen by
Neeraja et al. (2004) in poultry birds on maduramicin toxicity and by giving ginseng and carotene their occurrence was reduced. Degeneration and necrosis were reported in the poultry hearts by
Sadek et al. (2009) after salinomycin toxicity and addition of vitamin E or selenium diminished the severity of cardiac lesions. In our study also, vitamin C addition reduced the severity of heart lesions in intoxicated groups.
Skeletal muscles and gizzard
On gross examination, in group III and V, dryness and petechiae in the thigh muscles and breast muscles could be seen (Fig 1c). Pallor, wasting and loss of shine in muscles of birds of this group made them appear atrophied (Fig 1d) and especially ilio-tibialis was severely atrophied. Microscopically, there was severe degeneration and necrosis of skeletal muscles in group III and V characterized by widespread disruption and fragmentation of skeletal myofibers with loss of cross striations (Fig 2e, 2g). Expanding the interstitial space, eosinophilic fibrillar and karyorrhectic debris was seen interspersed between hyalinized myofibrillar fragments. Large aggregates of heterophilic infiltrates expanded perimysium and compressed eosinophilic degenerating and necrotic muscle fibers which were separated by interstitial edema (Fig 2e). In group IV and VI the muscles appeared tout with only mild intermuscular edema and myofiber degeneration (Fig 2f, 2h). In group III and V, gizzard musculature lacked normal glistening, seemed shrunken and atrophied. Histologically, examination of gizzard in ionophore alone administered groups revealed severe degeneration, necrosis of muscularis layer where the muscle fibers were replaced by fibrillar necrotic material and a heavy infiltration of inflammatory cells but mucosa was relatively spared. Intermuscular areas were expanded by dilated vessels with degenerating
tunica media and infiltrate encompassing their serosa, perivascular edema, heterophilic and macrophage infiltration and fibroblastic proliferation. (Fig 2i, 2k). No obvious gross changes were seen in gizzard muscles of group IV or VI group and histoarchitectural alterations were significantly less severe with only multifocal myo-degeneration predominantly vacuolation of sarcoplasm interspersed between healthy muscle fibers (Fig 2j, 2l). Extensive necrosis and fragmentation of muscle fibers in turkey birds due to accidental intake of salinomycin was seen by
Assen (2006).
Neeraja et al. (2004) also reported haemorrhage in thigh muscles after maduramicin toxicity.
Diaz et al. (2018) observed cellular necrosis of pectoral, ilio-tibialis and breast muscle of birds due to salinomycin toxicity. Pallor of muscles, severe necrotic and degenerative myopathy with infiltration of neutrophils and macrophages in the muscles were seen in rabbits by
Peixoto et al. (2009) because of intake of salinomycin. Likewise,
Chen et al. (2014) found that maduramicin toxicity in birds caused skeletal muscle degeneration. Hyper eosinophilia, fragmentation, loss of cross striations and degenerative changes in the skeletal muscles in lactating sow was described due to salinomycin and maduramicin toxicity (
Britzi et al. 2017). Similar to our results with vitamin C administration,
Sadek et al. 2009, also found significant improvements in salinomycin induced pathomorphological alterations after vitamin E or selenium supplementation.
Liver and kidneys
In group III, severe congestion and hemorrhages were seen. Severe vacuolar degeneration and necrosis of hepatocytes with focal infiltration of inflammatory cells predominantly lymphocytes were seen (Fig 2m). In group IV, only congestion and mild degenerative changes and occasional focal necrotic areas could be appreciated (Fig 2n). In group V, severe lesions were seen which included congestion, hemorrhage, degeneration and necrosis. Necrotic areas were infiltrated by lymphocytes. In portal triad area, severe inflammatory changes were seen which included congestion, edema and inflammation (Fig 2o). In group VI, less severe changes were seen which included mild degeneration and congestion (Fig 2p). As far as kidneys were concerned, in group III birds there was tubular vacuolar degeneration along with focal necrosis of tubules. In the inter tubular areas in some birds, there was severe hemorrhage, congestion and edema (Fig 2q). In many areas, frank necrosis of tubules with hemorrhage and infiltration by heterophils was observed. In group IV, less severe changes consisting mostly of mild degeneration were seen. (Fig 2r). In group V birds, changes consisted of severe degeneration, necrosis of tubules and heterophilic infiltration (Fig 2s). In group VI, mild degenerative changes were seen in addition to mild congestion (Fig 2t). Similar changes in liver like enlargement, yellow discoloration and degeneration were also seen by
Neeraja et al. (2004) after experimental maduramicin toxicity in birds. They also noticed degenerative changes in kidneys. Addition of the ginseng and carotene ameliorated these lesions.
Rizvi et al. (2008) reported congestion of liver sinusoids and vacuolization of hepatocytes in broiler birds after experimental salinomycin toxicity.
Koutoulis et al. (2013) found liver degeneration and swollen kidneys in broiler birds due to salinomycin toxicity. In camels, salinomycin toxicity led to hepatomegaly with multifocal hemorrhage and necrosis of hepatocytes and degeneration in renal tubules (
Al-Wabel., 2012) which was similar to the findings of the present study.
Ashrafihelan et al. (2014) reported focal necrosis, retention of bile and cholangitis in the liver and pale kidneys with extensive acute tubular necrosis in sheep due to accidental intoxication of salinomycin. In zebra fish, maduramicin toxicity caused vacuolar degeneration in the liver
(Ni et al., 2019). Sadek et al. (2009) found degeneration and necrosis in liver and kidney in birds intoxicated with salinomycin and usage of vitamin E or selenium reduced the degenerative lesions. In the present study, vitamin C was used to counteract effects of ionophore induced toxic changes and reduced the pathological changes in kidneys and
Wong (2019) have also demonstrated that baicalein ameliorates cadmium induced oxidative hepato-renal damage.
Spleen
In group III, there was marked lymphocytic depletion in and around splenic arteriole and necrosis was also observed (Fig 2u). In group V, also lymphoid depletion and congestion was very pronounced (Fig 2w). Additionally, periarteriolar edema was observed. In contrast, in group IV and VI, lymphoid depletion was mild and no edema was seen (Fig 2v, Fig 2x). Similar findings have also been reported by
Koutoulis et al. (2013), who saw splenic atrophy in broiler breeders and turkeys because of salinomycin toxicity.
