The present study estimated the level of some macro and micro minerals in female dogs with mammary

gland tumors fed with different habitual diets. Female dogs bearing malignant mammary tumors were divided into two

groups; group-I (n=12) fed only with balanced commercial diet and group-II (n=12) fed only with homemade diet. Ten

physiologically healthy female dogs were taken as control. Trace elements viz. iron, copper, zinc and manganese were

estimated in plasma and mammary tumor tissues. Calcium and phosphorus were exclusively estimated in plasma. Plasma

concentrations of iron, copper and calcium were found to be significantly (p<0.05) elevated in homemade diet group as

compared to control. However, the level of zinc, manganese and phosphorus in plasma of female dogs fed only with

homemade diet was decreased (p<0.05) in comparison to control. The mammary gland tissue analysis of iron, copper, zinc

and manganese has revealed that their level was found to be significantly (p<0.05) increased in mammary gland tumor

tissues of both commercial and homemade diet group as compared to normal mammary tissues. The study concluded

that, homemade diet significantly influence the plasma mineral status in the tumor bearing female dogs. However, mineral

levels in mammary tumor tissues were altered irrespective of diet types

Chanchal Singh

Gurpreet Singh

Subrat Kumar Dash

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59Research  ArticleMINERAL STATUS IN FEMALE DOGS WITH MALIGNANT MAMMARY GLAND TUMORSFED WITH DIFFERENT HABITUAL DIETSSubrat Kumar Dash1, Chanchal Singh2*, Gurpreet Singh2Received 06 February 2018, revised 05 May 20181Ph D scholar, 2 Assistant Professor, Department of Veterinary Physiology and Biochemistry, College of Veterinary Science,Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, Punjab, India.*Corresponding author. e - mail: drchanchal833@gmail.comABSTRACT: The present study estimated the level of some macro and micro minerals in female dogs with mammarygland tumors fed with different habitual diets. Female dogs bearing malignant mammary tumors were divided into twogroups; group-I (n=12) fed only with balanced commercial diet and group-II (n=12) fed only with homemade diet. Tenphysiologically healthy female dogs were taken as control. Trace elements viz. iron, copper, zinc and manganese wereestimated in plasma and mammary tumor tissues. Calcium and phosphorus were exclusively estimated in plasma. Plasmaconcentrations of iron, copper and calcium were found to be significantly (p<0.05) elevated in homemade diet group ascompared to control. However, the level of zinc, manganese and phosphorus in plasma of female dogs fed only withhomemade diet was decreased (p<0.05) in comparison to control. The mammary gland tissue analysis of iron, copper, zincand manganese has revealed that their level was found to be significantly (p<0.05) increased in mammary gland tumortissues of both commercial and homemade diet group as compared to normal mammary tissues. The study concludedthat, homemade diet significantly influence the plasma mineral status in the tumor bearing female dogs. However, minerallevels in mammary tumor tissues were altered irrespective of diet types.Key words: Female dogs, mammary tumors, minerals, diet.INTRODUCTIONMammary gland tumors are most commonly diagnosedneoplasia in female dogs. It is second in line after skincancer in canines (Kumaraguruparan et al. 2005). Theincidence of the disease is increasing in canine population.The increasing prevalence of mammary tumours has beenattributed to change in habitat and dietary pattern offemale dogs from free ranging to the home made diet asconsumed by human (Karayannopoulou et al. 2013). Theminerals present in diet viz. iron, copper, zinc, manganese,calcium, phosphorus etc. are derived from the naturalsources from which the diet is prepared and influencedby mineral content of water, soil and plants (Becker etal. 2007). Hence deficiency or excess of minerals in thenatural source is reflected in the food. The commonmisconception among people is that by supplementinghomemade diet, they are feeding a healthy diet to theirbitches; however, key minerals may be missing or arefed in excess through homemade diet. Homemade foodis not necessary for good health of female dogs ratherfood with appropriate proportion of nutrients is therequirement (Karayannopoulou et al. 2013).Somedeficiency and excess has been so severe that mineralconcentrations did not reach 50% of the NationalResearch Council (NRC) recommendations or are morethan the maximum limit suggested by NRC respectively(Perez et al. 2000). Hence, dietary control of theseminerals is important with respect to canine health anddisease. It is important that during health problem likecancer, diet of female dogs must “complete andbalanced,” means it should meet all of the dog’snutritional needs which can be achieved by feedingcommercially available balanced canine food(Karayannopoulou et al. 2013). Commercial pet foodsare made from a fixed formula of ingredients andpreparation methods. These foods are supplemented withappropriate proportion of canine grade minerals andvitamins required for dogs providing them a balanceddiet (Perez et al. 2000).Mammary cancers like any otherdiseases are associated with imbalanced mineral statusExplor Anim Med Res,Vol. 8, Issue - 1, 2018, p. 59-63ISSN  2277- 470X (Print), ISSN 2319-247X (Online)Website: www.animalmedicalresearch.org60Exploratory Animal and Medical Research, Vol.8, Issue 1, June, 2018which is reflected in their plasma and tissueconcentrations (Arinola and Charles 2008).Determinations of the plasma and tissue level of mineralsin tumour bearing patients provide their status in thediseased state which will help in better diagnosis andprognosis.The study evaluated the concentration of someminerals in female dogs with malignant mammary glandtumors fed with different habitual diets.MATERIALS  AND  METHODSAnimalsFemale dogs (aged 6-14 years) bearing malignantmammary gland tumors were selected from the casespresented at Small Animal Clinical Complex, Guru AngadDev Veterinary and Animal Sciences University,Ludhiana. These animals were clinically examined anddiagnosed for malignant mammary tumor (based onhistopathology, radiography and ultrasonography) by theDepartment of Veterinary Surgery and Radiology.Clinically healthy female dogs presented at the clinicsfor routine checkup were taken as control. The animalswere divided into two groups:Control (n=10): Clinically healthy female dogsGroup-I (n=12):  fed only with balanced commercialdiet andGroup-II (n=12): fed only with homemade diet.Dietary historyBased on a questionnaire, the owners were asked aboutthe diet over past years. Focus was given particularly tothe dietary regimen in the months preceding the diagnosis.Approximate date of starting of diet was also recorded.The dogs were fed twice daily; morning and evening.Dogs were supplemented with homemade diets usuallyrich in minerals (calcium, iron, copper, zinc andmanganese) that consisted of butter, egg, milk, fish,mineral rich vegetables, fruits and specific foods likeSimple carcinoma Squamous cell carcinoma Carcino-sarcoma2 6 42 7 3Commercial groupHomemade groupDiet groups Tumor types (n)n= number of cases.Table 1. Types of malignant mammary tumors observed in the study.Minerals Control Group-I Group-IICalcium (mg/dL) 9.68±0.14 9.85±0.22 14.56±0.44*Phosphorus (mg/dL) 4.73±0.07 4.52±0.07 3.51±0.08*Iron (µg/dL) 87.25±2.06 88.31±2.11 167.61±3.02*Copper (µg/dL) 105.11±2.12 106.04±2.52 191.67±2.88*Zinc (µg/dL) 64.23±1.04 65.71±2.01 36.71±2.08*Manganese (µg/dL) 17.25±0.22 17.69±0.35 12.61±0.33** Significantly (p<0.05) differ from the control.Table 2. Concentration (Mean ± SE) of macro and micro minerals in plasma of bitches with mammary tumors.Micro minerals Normal mammary Group-I Group-IItissue (Control)Iron (µg/g) 142.28±1.98 178.77±3.66* 257.08±3.18*Copper (µg/g) 12.32±0.95 20.18±0.93* 28.05±1.02*Zinc (µg/g) 24.19±0.92 27.55±1.18* 29.37±1.74*Manganese (µg/g) 2.87±0.09 3.91±0.25* 5.18±0.22** Significantly (p<0.05) differ from the control.Table 3. Concentration (Mean ± SE) of micro minerals in mammary tumor tissues of bitches.61chicken and red meat (pork or lamb). Diet was preparedby the traditional method of boiling and in maximumcases butter or palm olive oil was used for cooking thesefoods. Commercial diet consisted of balanced canned,semi-moist or dry dog food. The brand of canned foodwas recorded. As mentioned in the label of commercialfoods, it consisted of approximately meat or meat derivedproducts (30%), oils or fats (10%), cereals and vegetables(5%) along with required quantity of vitamins andminerals.Collection and analysis of samplesBloodFour mililitres of blood were collected in heparinisedcentrifuge tubes from the cephalic vein of healthy (n=10)and tumor bearing female dogs; commercial (n=12) andhomemade (n=12) diet groups. Blood samples werecentrifuged at 2500 rpm for 10 minutes and plasma wasseparated.Mammary tissuesFive grams of fresh mammary tumor tissues werecollected both from commercial (n=12) and homemade(n=12) diet groups of female dogs immediately aftersurgery. Adjacent normal mammary tissues (n=10)collected from these female dogs were taken as control.HistopathologySmall portions of tissues were fixed in 10% neutralbuffered formalin (NBF), embedded in paraffin and tissuesections were prepared. The tissue sections were stainedwith Haematoxylin and Eosine (H & E) for microscopicexamination.Estimation of macro mineralsCalcium and phosphorus were determined in plasmausing Autopak kits (Siemens Healthcare Diagnostics Ltd.)on semiautomatic biochemical analyzer. All thedeterminations were performed in duplicate.Estimation of micro mineralsPlasma (2ml) and mammary tissue (3g) samples wereigested after adding 15 ml of tri-acid mixture (HNO3,H2SO4 and HClO4 in 10:4:1 ratio) until a transparentsolution was obtained (Ludmila 1976). After cooling, theigested samples were filtered and the volume was madeto 10 ml with doubled distilled water. Iron Copper, Zincand Manganese concentration in digested plasma andmammary tissue samples were estimated using atomicabsorption spectrophotometer (AAS 4139, ElectronicsCorporation of India Limited, Hyderabad).Statistical analysisData were subjected to analysis of variance (ANOVA)using statistical package for Social Sciences (SPSS)software (Version 16.0). The results were expressed asmean ± SE of mean (SEM). Correlation between differentparameters was determined by Karl Pearson’s correlationcoefficient.RESULTS  AND  DISCUSSIONHistopathological analysis of tumor tissue samplesrevealed that both commercial and homemade diet groupsof female dogs were having simple carcinoma,adenocarcinoma and carcino-sarcoma of mammary gland(Table 1). In the homemade diet group, plasmaconcentration of calcium, iron and copper has been foundto be significantly (p<0.05) increased compared tocontrol. This was accompanied by significant (p<0.05)decline in plasma level of phosphorus, zinc andmanganese in the homemade diet group (Table 2). Foodprepared in home may be treated as a balance diet forhuman; but may not be balanced in all respect for canines(Karayannopoulou et al. 2013). Minerals like zinc,copper, iron, manganese, calcium and phosphorus maybe deficient or excess in the homemade diet dependingon the type of diet and geographical region from whichMineralsIron (Plasma) Vs. Iron (Tumor tissue)Copper (Plasma) Vs. Copper (Tumor tissue)Zinc (Plasma) Vs. Zinc (Tumor tissue)Manganese (Plasma) Vs. Manganese (Tumor tissue)Pearson’scorrelationcoefficient (r)0.22*0.18*0.12*0.11**Indicates significant difference at p<0.05.Table 4. Correlation between concentration of microminerals in plasma and mammary tumor tissues of bitchesfed with commercial diet.MineralsIron (Plasma) Vs. Iron (Tumor tissue)Copper (Plasma) Vs. Copper (Tumor tissue)Zinc (Plasma) Vs. Zinc (Tumor tissue)Manganese (Plasma) Vs. Manganese (Tumor tissue)0.71*0.64*-0.55*-0.57*Pearson’scorrelationcoefficient (r)*Indicates significant difference at p<0.05.Table 5. Correlation between concentration of microminerals in plasma and mammary tumor tissues of bitchesfed with homemade diet.Mineral status in female dogs with malignant mammary gland tumours fed with different habitual diets62Exploratory Animal and Medical Research, Vol.8, Issue 1, June, 2018the raw materials used for preparation of these diets werederived (Arinola and Charles 2008).Female dogs fed with unbalanced homemade diet havebeen observed to have compromised health (Vannucchiet al. 2007). Calcium, iron, copper and phosphorus areessential for numerous biochemical processes in the cell.However, excess serum calcium with low phosphoruslevel is associated with different types of malignancies(Nathaniel et al. 2010). Hypercalcemia andhypophosphatemia observed in mammary tumour patientscan be attributed to enhancement in bone osteolysis anddisturbances in the reabsorption and excretion of calciumand phosphate ions (Esbrit 2001). The most commoncancer associated with high calcium and low phosphorusis mammary cancer (Nathaniel t al. 2010) which is inagreement with our study. Increased plasma levels ofcalcium, iron and copper observed in the homemade dietgroup might be a consequence of intake of more butter,milk, fish and vegetables in diet. Zinc and manganeseare required for proper growth and division of cells.However, neoplastic cells can also use these elementsfor their proliferation (Smith et al. 2007). Lowered levelof plasma zinc and manganese in tumour bearing femaledogs might be due to the consumption of these elementsby the malignant mammary tissues for their growth andproliferation (Arinola and Charles 2008). In our studylevel of copper, iron, zinc and manganese were elevatedin mammary tumor tissues of both homemade andcommercial diet groups (Table 3). Copper and iron areessential elements for health. These elements serve ascofactors of the enzyme superoxide dismutase However,excess iron and copper can be toxic and generate reactiveoxygen species through Fenton reaction causing oxidativestress (Kumaraguruparan et al. 2005). Studies suggestedthat antioxidant enzymes are over expressed in tumortissues to provide protection against the oxidative stress(Kumaraguruparan et al. 2005, Filomeni et al. 2007).Elevated levels of iron, copper, zinc and manganese inmammary tumor tissues of both homemade andcommercial diet groups might be due to increasedexpression of superoxide dismutase enzyme and uptakeof these elements from the plasma by the tumour cellsfor their proliferation and conversion into a malignantphenotype (Arinola and Charles 2008) which has beensupported by the significant correlations observedbetween their plasma and tumour tissue levels in bothcommercial diet (Table 4) and homemade (Table 5) dietgroups. Previously conducted studies suggested thatfemale dogs bearing mammary tumors fed withhomemade diet were observed to be associated withimbalanced plasma and tissue minerals (Perez et al. 1998,P rez et al. 2000 and Karayannopoulou et al. 2013) whichis in line with the results of the present study.CONCLUSIONThe study concluded that, homemade diet significantlyinfluence the plasma mineral status in the tumor bearingfemale dogs. However, mineral levels in mammary tumortissues were altered irrespective of diet types.ACKNOWLEDGEMENTAuthors are thankful to the Director of Research, GuruAngad Dev Veterinary and Animal Sciences University,Ludhiana for providing funds for this study.REFERENCESArinola  OG, Charles MA (2008) Micronutrient level inplasma of Nigerian females with breast cancer. Afr J Biotec.7(11): 1620-1623.Becker JS, Matusch A, Depboylu C, Dobrowolska J, ZoriyMV (2007) Quantitative imaging of selenium, copper, and zincin thin sections of biological tissues (slugs-genus Arion)measured by laser ablation inductively coupled plasma massspectrometry. Anal Che. 79(16): 6074-6080.Esbrit P (2001) Hypercalcemia of malignancy: New insightsinto an old syndrome. Clin Lab  47 (1-2): 67-71.Filomeni G, Cerchiaro G, Da Costa Ferreira AM, De MartinoA, Pedersen JZ, Rotilio G, Ciriolo MR (2007) Pro-apoptoticactivity ofnovel Isatin-Schiff base copper (II) complexesdepends on oxidativestress induction and organelle-selectivedamage. J Biol Chem  282(16): 12010-12021.Kumaraguruparan R, Balachandran C, Manohar BM, NaginiS (2005) Altered oxidant-antioxidant profile in canine mammarytumours.Vet Res Commun  29(4): 287-296.Karayannopoulou M, Fytianou A, Assaloumidis N, PsallaD, Savvas I,  Kaldrymidou E (2013) Lipid peroxidation inneoplastic tissue of dogs with mammary cancer fed withdifferent kinds of diet.Turk  J Vet Anim Sci  37: 449-453.Ludmila D (1976) Chemical analysis by atomic absorptionspectroscopy. Varian Techtron Pvt Ltd, Melbourn, Australia.Nathaniel I, Maxwell C, Omabbe Chinyere AO, Augusta N(2010) Calcium, inorganic phosphates, alkaline and acidphosphatase activity in breast cancer patients in Calabar,Nigeria. Afr Health Sc  10(1): 9-13.63Perez AD, Rutteman GR, Pena L, Beynen AC, Cuesta P(1998) Relation between habitual diet and canine mammarytumors in a case-control study J Vet Intern Med  12: 132-139.Perez AMD, Pena L, Del CN, Nieto AI (2000) Factorsinfluencing the incidence and prognosis of canine mammarytumours. J Small Anim Pract  41(7): 287-291.*Cite this article as: Dash SK, Singh C, Singh G (2018) Mineral status in female dogs with malignant mammary gland tumors fedwith different habitual diets. Explor Anim Med Res  8(1): 59-63.Smith D, Gwiazda R, Bowler R, Roels H, Park R, TaicherC, Lucchini R (2007) Biomarkers of Mn exposure in humans.Am J Ind Med  50(11): 801-811.Vannucchi CI, Jordao AA, Vannucchi H (2007) Antioxidantcompounds and oxidative stress in female dogs duringpregnancy. Res Vet Sci  83:188-193.Mineral status in female dogs with malignant mammary gland tumours fed with different habitual diets

MINERAL STATUS IN FEMALE DOGS WITH MALIGNANT MAMMARY GLAND TUMORS FED WITH DIFFERENT HABITUAL DIETS

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MINERAL STATUS IN FEMALE DOGS WITH MALIGNANT MAMMARY GLAND TUMORS FED WITH DIFFERENT HABITUAL DIETS

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