Golden mussel shell ( Limnoperna fortunei ) flour contaminated with cadmium as a calcium source for broiler chickens

The objective of this study was to evaluate the effects of golden mussel flour (GMSF) contaminated with increasing rates of cadmium (Cd) as a replacement for limestone as a Ca source, in broiler chicken feed from 14 to 42 days of age. A total of 60 animals were assigned to four treatments (inclusion rates of Cd: 6.94, 14.55, 22.40, and 30.00 mg Cd kg -¹ ) with five replications in a completely randomized design. At 42 d, blood samples were collected to evaluate serum concentrations of aspartate amino transferase (AST), alanine amino transferase (ALT), gamma glutamyl transferase (GGT), total bilirubin, Ca, P, and Cd. After slaughter, tissues were collected to evaluate Cd concentration in bone parameters. Growth performance of broiler chickens and Cd content in the breast meat were not affected by the inclusion rates of Cd in the GMSF. However, there was an effect of Cd in GMSF on the concentration of Cd in the skin, liver, bones, feathers, and serum; ALT; and total bilirubin. Bone flexibility had a quadratic response to increasing inclusion rates of GMSF; serum Ca concentration increased linearly and there was no effect on serum P concentration. Concentrations of Cd in GMSF above 20 mg kg -¹ caused high Cd contamination in broiler tissues. Therefore, it was concluded that Cd concentrations above 6.94 mg kg-¹ in broiler diets caused high Cd concentrations in meat and organs that are above those permitted for human consumption.


Introduction
The demand for animal protein by the population places constant pressure on the meat production sectors, such as the poultry sector (Xu et al., 2015;Bayerle et al., 2017).However, this increase in the production of broilers results in an increased need for raw materials to feed the animals.This requirement has driven the research for new food sources that replace or can be used as effective ingredients within the nutritional programs.In this context, the by-products of the food industries become the main alternatives and sources for this purpose (Nunes et al.,2018).
The use of by-products in animal diets can reduce the incorrect disposal of these products into the environment (Kar & Patra, 2021).When used in broiler diets, these alternative ingredients can reduce production costs, resulting in more economically viable and nutritionally productive feed (Nunes et al., 2018;Olgun et al., 2020).
Golden mussel flour can be an economic, nutritional, and environmentally acceptable alternative, especially in regions where its occurrence is unwanted and its acquisition is low-cost.The use of the golden mussel (Limnoperna fortunei) as a source of Ca in animal feed is based on its low cost and the fact that this bivalve mollusc is an invasive, exotic species that causes economic and environmental damage (Wachholz et al., 2017).Thus, its use in the animal industry would reduce environmental problems of obstruction of the passage of water in pipes, channels and collecting systems of water, filters, pumps, and refrigeration systems (Akter et al., 2019).
However, the main obstacle to using this mollusc in animal feeding is the filtering habit of this species, which can lead to the absorption of pollutants from the waters where it lives.Among these pollutants are toxic metals, including cadmium (Cd), which is an environmental pollutant (Marengoni et al., 2013), hazardous to humans and animals (Olgun et al., 2015;Zwolak, 2020) with potential carcinogenic effects.Cadmium exposure results in bioaccumulation of this element in different tissues, particularly in the liver, kidney, brain, pancreas, intestine, and reproductive organs, which increases oxidative stress at cellular levels due to the overproduction of reactive oxygen species (Zhang et al., 2018).These phenomena induce dysfunctions of biologically important cellular molecules, resulting in various gross and histopathological changes in these organs and haemato-biochemical alterations.Consequently, reductions in growth performance can occur in poultry (Kar et al., 2018;Akter et al., 2019;Olgun et al., 2020).Exposure of livestock including poultry to Cd not only affects health, but also hampers animal production by reducing growth performance and feed efficiency (Olgun et al., 2020).Studies have shown that Cd has negative effects on growth performance (Al-Waeli et al., 2013;Olgun & Bahtiyarca 2015), while others reported positive effects using low dietary levels (Cigankova et al., 2009;Olgun, 2015).The blood profile can also be negatively affected by Cd.Some research has reported liver lesions (Cinar et al., 2010;Ali et al., 2016;Tahir et al., 2017;Gutyj et al., 2019) and changes in plasma concentrations of alanine aminotransferase (ALT), triglycerides, and glucose (Ali & Abdulla 2013;Kar & Patra, 2021).Age and species of bird are also factors that can cause variations in the level of ingested Cd, which is toxic when absorbed by the organism.In addition, Cd uptake toxicity can vary according to cell type in the kidneys and liver (Li et al., 2018;Olgun et al., 2020).Nad et al. (2012) evaluated Cd absorption in turkeys and observed an intense accumulation of Cd in bones, muscles, liver, and kidneys.Similar results have been reported by other studies (Ali et al., 2016;Jin et al., 2018;Kar et al., 2018;Vasiljeva et al., 2018), suggesting that liver and kidneys are more sensitive to Cd accumulation.
Thus, it is necessary to conduct studies to verify the amount of Cd in golden mussel shells in broiler chickens can utilize without affecting performance or causing tissue contamination (Wachholz et al., 2017).In addition, these studies may be useful to determine the potential risks of this heavy metal, as well as its accumulation in tissues, to provide information to local regulatory authorities for future recommendations of maximum allowable values (Khan et al., 2016, Huang et al., 2019, Jawad et al., 2021).
The objective of this study was to evaluate the use of golden mussel shell flour contaminated with different inclusions of Cd in the diet of broiler chickens from 14 to 42 days of age and determine its effects on growth performance, tissue contamination, Cd digestibility, bone quality, and blood metabolites.

Materials and Methods
This study was conducted at the Poultry Research Centre of Western Paraná State University (Marechal Cândido Rondon, PR, BR).All the procedures were performed according to the National Council for Control and Animal Experimentation and approved by the Animal Use Ethics Committee of the university under the protocol number 04/2017.
Golden mussel shells were collected on the banks of the reservoir from ITAIPU Hydroelectric Power Plant, (Marechal Cândido Rondon -PR, BR), dried by sun exposure, and milled in a hammer mill (4-mm sieve).Golden mussel shell flour (GMSF) was sampled and analysed for the contents of Cd, P, and Ca using atomic absorption spectrometry and its composition was 6.94 mg of Cd kg -¹ , 3.98 mg P kg -¹ , and 30.64 mg Ca kg -¹ .
From days 1 to 14, broiler chickens were raised in an aviary with concrete floor covered with pine wood shavings and received a common initial ration formulated according to the recommendations of Rostagno et al. (2011).A total of 60 one-day old male broiler chickens (Cobb 500, Cobb-Vantress Ltd., Cascavel, PR, BR) vaccinated for Marek, Gumboro, fowl pox, and infectious bronchitis, with an initial mean weight of 393.95 ± 14.12 g, were housed in cages of 50 cm², with three broiler chickens per experimental unit (cage).The experimental design was completely randomized, with four treatments (inclusion rates of Cd in the flour of mussel shells) and five replications of three broiler chickens per treatment.Mussel shells were used as a Ca source and the concentrations of Cd above 6.94 mg kg -¹ were obtained by adding Cd nitrate [Cd(NO3) 2 .4(H2O)] to the GMSF.The four treatments were increasing inclusions of Cd in GMSF: 6.94 mg Cd kg -¹ of GMSF (without induced contamination), 14.55 mg Cd kg -¹ of GMSF, 22.40 mg Cd kg -¹ of GMSF, and 30 mg Cd kg -¹ of GMSF in the starter (14 to 28 d) and grower phases (29 to 42 d).Two experimental diets were used according to the rearing phase of broiler chickens (grower and finisher phases), and the GMSF was the main Ca source in the diets.Broiler chickens received diets to meet the nutritional requirements proposed by Rostagno et al. (2011) (Table 1) for males during the grower (14 to 28 d) and finisher (29 to 42 d) phases, with feed and water provided ad libitum throughout the experiment.Mineral supplement (g/kg of diet): copper 0.09 g, iron 0.06 g, manganese 0.04 g, cobalt 1 mg, iodine 1 mg, zinc 0.17 g; 3 butylated hydroxytoluene (BHT); 5 salinomycin 4% Weight gain (WG), feed intake (FI), and feed conversion ratio (FCR) were recorded from day 1 to day 42.Mean individual bird weight and FI were calculated, taking mortalities into consideration (Sakomura & Rostagno, 2016).At 42 days, two birds per pen were randomly selected, fasted for six hours, and blood samples were collected via brachial puncture.Blood was coagulated and centrifuged at 1008 × g for 10 min to obtain serum, which was stored at -20 °C.To perform the analyses, serum was thawed at room temperature, centrifuged at 1008 × g for 5 min, and then aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transferase (GGT), total bilirubin, Ca, and P analyses were performed with a high-performance automatic spectrophotometer (Flexor EL 200, Elitech, Paris, France) with specific kits, calibrated with the appropriate standards (Elical, Elitech, Paris, France).After blood collection, three birds were weighed and euthanized by electrocution followed by exsanguination (Normative Resolution No. 37 of February 15, 2018, Concea) for skin collection (around the chest), muscle (pectoralis major), breast, feathers, bones (femur), and liver to determine the Cd content.
Total excreta collection (Sibbald & Slinger, 1963) was performed from 38-42 d twice a day with an interval of 12 h to minimize fermentation.During this collection period, trays were covered with plastic and placed under each cage to avoid losses and contamination.The material collected was packed in plastic bags according to the experimental unit, duly weighed, and stored in a freezer (-20 °C) until analysis.
For bone quality evaluations, meat from the tibia was removed and thereafter, the stiffness and bone-breaking strength were measured using a CT3 texture analyser (CT3 Texture Analyzer, Brookfield Engineering Laboratories, Inc., Middleboro, MA, US) by applying 200 kgf at a speed of 5 mm/s in the central bone region (diaphysis); the values were expressed as mm and kgf.Bone flexibility is expressed in kgf/cm and calculated from breaking strength and stiffness.
The concentration of Cd in feed and excreta (mg kg -¹ ) were determined using an indigestibility factor (IF), which was obtained from the acid insoluble ash (AIA).Apparent metabolizable Cd (AMCd) was obtained according to Sakomura & Rostagno (2007) and the apparent digestibility coefficient of Cd (ADCCd) was adapted from Rostagno & Featherston (1977).Data were expressed on a dry matter basis and calculated according to the following formulae: (2) Data were subjected to analysis of variance at 5% probability level and subsequent polynomial regression for the inclusion rates of GMSF.Statistical analyses were performed using SAS software (SAS Inst.Inc., Cary, NC, US) in a completely randomized design, considering the cages as the experimental units.

Results and Discussion
The contamination with cadmium nitrate -Cd(NO3) 2in the GMSF was not enough to cause toxicity to the animals, nor reduce its growth performance (Table 2).This result was probably related to the low inclusion of Cd in the GMSF.In the study by Olgun et al. (2020), it was observed that cadmium concentrations of 10-20% provide toxicity and reduce the performance of cutting chickens up to 45 days of age.Lisunova & Tokarev (2016) observed that the inclusion of 0.002 mg Cd kg -¹ in diets was sufficient to cause a 6.2% reduction in weight gain in cutting chickens up to 49 days of age.
In the study of Al-Waeli et al. (2013) with broiler chickens fed diets with 10 mg kg -¹ Cd, negative effects on growth performance and mortality were not observed.However, when 100 mg kg -¹ Cd per unit body mass was offered, feed intake decreased and feed conversion increased.These observations suggest that Cd, besides causing metabolic and pathological changes in different organs, can directly impair nutrient digestion and absorption in the gut, resulting in poor feed conversion ratio and production performance of poultry (Kar & Patra, 2021).There were no effects of Cd inclusion on its concentration in the pectoralis major muscle (Table 3).However, the Cd concentration used for all the diets evaluated was above the recommended by FAO (2000) (0.05 mg kg -¹ ).
Studies have shown that Cd accumulates preferentially in the inner organs and in the liver and kidneys (Kurnaz & Filazi, 2011;Karimi et al., 2017).However, in the present study, a high Cd concentration was also found in the skin.Cadmium concentration was higher in serum (2.95 mg kg -¹ ) followed by bone, feathers, skin, liver, and muscle.Zhuang et al. (2014) evaluated broiler chickens fed with metal-enriched rice (0.24 mg Cd kg -¹ ) and obtained a higher content of Cd in the liver (9.36 mg kg - ¹ ) followed by kidney, feather, muscle, and blood (4.64, 0.51, 0.059, and 0.042 mg kg -¹ of dry weight, respectively).Cinar et al. (2010) evaluated lower doses of Cd, and no such remarkable changes were found in chicks.However, severe hydropic degeneration was reported in liver tissues with Cd administration (60 mg kg -¹ ) in a basal diet of broiler chickens for 42 d.(2016) showed that daily administration of Cd (50 mg L -¹ in drinking water) for 45 d to broiler chickens induced degenerative changes in the hepatocytes; increases in sinusoidal spaces; and swollen, fragile, and focal necrotic spots in livers.Gabol et al. (2014) investigated the effect of Cd at various doses (low dose of 10 μg kg -¹ body weight and a higher dose of 20 μg kg -¹ body weight) and observed that the higher doses of Cd caused increased hepatic cell size, damage and necrosis of the hepatic cells, and infiltrations of numerous macrophages in the liver.In addition, cadmium induces alterations in normal liver function, causing a wide range of alterations in blood profiles (Akter et al., 2019;Ali et al., 2021;Jawad et al., 2021;Kar & Patra, 2021).Gutyj et al. (2019) reported that Cd (2 and 4 mg kg -¹ body weight) for white laying hens (78 weeks of age) for 30 d resulted in deviations from normal values of blood biochemical profile due to pathological changes in the organs.As Cd causes extensive pathological changes in different vital organs, particularly in the liver, kidney, lungs, and reproductive organs, these alterations are reflected in the blood metabolites.
Table 4 Blood metabolites of broiler chickens fed with golden mussel shell flour contaminated with increasing inclusions of Cd Cd (mg kg -¹ ) 1 AST (U L -¹ ) 2 ALT (U L -¹ ) 3 GGT (IU L -¹ ) Total Bilirubin (mg dL The minimum estimated value of ALT (5.454 mg Cd kg -¹ ) observed in the present study corroborates the value reported by Adaramoye & Akanni (2016) in a study with rats, where Cd contamination caused changes in the ALT activity.When the circulatory Cd is greater than the binding capacity of metallothionein, the free Cd induces free radicals and lipid peroxidases and can damage kidneys and liver (Gałazyn-Sidorczuk et al., 2009;Heshmati & Salaramoli, 2015;Korénekova et al., 2017;Olgun et al., 2020).An increased Cd concentration in serum may be related to the higher activity of the enzyme, ALT, at higher inclusions, since Cd can induce injuries in the liver and kidney due to its ability to enhance free radical formation in vivo (Abdo & Abdulla, 2013;Gutyj et al., 2019).Ali et al. (2016) used 10 mg Cd kg -¹ of diet for 4 w in the diet of broiler chickens and found an increased activity of ALT and AST.When compared to the control group, the authors attributed this effect to hepatocellular damage or cellular degradation by this heavy metal, possibly in the liver, but in the heart or muscle.Karimi et al. (2017) evaluated Cd in the diets of Japanese quail with 100 mg kg -¹ for 60 d and obtained an increase in ALT activity.Similarly, the same group of researchers in two experiments with male Japanese quail reported that administration of Cd at 25 mg kg -¹ of feed (in both experiments) increased total serum protein and ALT activity (Karimi et al., 2015;2016).Corroborating our results, it is clear that Cd increases liver enzyme activities.The increase in serum bilirubin is associated with fat digestion and absorption (Andjelkovic et al., 2019).Silva et al. (2007) obtained total bilirubin values of 0.41 mg dL -¹ , similar to those obtained in the present study (0.45 mg dl -¹ ).
According to Oliveira et al. (2014), resistance and flexibility are the most important factors in cases of bone fractures.Liao et al. (2017) evaluated Cd in the diet of ducks and obtained a reduction in bone quality; studies evaluating Cd contamination in humans also found a reduction in bone quality (Chen et al., 2014;Birr et al., 2015).The serum concentration of P was not influenced by the inclusion of GMSF contaminated with Cd in the diet (Table 5).However, the concentration of Ca increased linearly with Cd inclusion in the diet.Cadmium can affect bone health, reducing the absorption of Ca from the intestines, increasing its excretion from kidneys, and preventing Ca incorporation and collagen production in bone cells (Wachholz et al., 2019).In addition, minerals such as P and Mg are affected by Cd in animals (Olgun et al., 2015) and can affect the biomechanical properties of bone, such as resistance to breaking (Olgun et al., 2020).In the present study, only flexibility was influenced (quadratically), and the values obtained with the increased inclusion rates of Cd were above those obtained for the basal diet.The bone parameters, deformation and breaking strength were not altered by the inclusion of Cd in the GMSF (Table 6).Corroborating that, the study of Olgun et al. (2015) evaluated increasing inclusion rates of Cd (5, 15, and 45 mg kg-¹) for laying hens and found no effects on Ca, P, and Zn content in the tibia of laying hens.However, the authors observed that higher doses of cadmium (>10 mg kg -¹ ) lead to toxicity symptoms, worsening productive performance in poultry.The apparent metabolizable Cd increased linearly (P <0.05) as well as the coefficient of apparent digestibility (CAD) of Cd (P<0.05)(Table 7).Apparent metabolizable Cd increased (AMCd) and its metabolizable coefficient increased with Cd inclusion.After absorption, this metal is excreted in urine, faeces, and bile, but has a low excretion rate (Suttle, 2010).According to ATSDR (2007), the estimated absorption rate of Cd by oral exposure for humans may vary from 1.1-10.6%.Therefore, a tolerable monthly Cd intake of 25 μg kg -¹ body weight (Chen et al., 2021) may occur in some industrial areas where human inhabitants may be vulnerable to Cd exposure through foods.In this context, with advances in Brazilian poultry farming, concern about the biosafety of ingredients used in the diets of farm animals has increased in recent years (Bayerle et al., 2017).Contamination of poultry feed is a problem and the scientific community strives to find solutions to increase food quality and safety (Wachholz et al., 2019).Heavy metals such as Cd are toxic agents involved in several disorders of animals and humans and limits in rations must be constantly updated (Wolf & Cappai, 2021).Distribution of Cd driven from feed in target organs is a key for estimating health risk from this exposure and determines the safety of animal products.In general, Cd can influence the absorption of Ca, as Cd uses the same transporters as Ca in animals (Reeves & Chaney, 2008).However, it is not possible to completely eliminate the damage in the liver and kidney and the injuries caused by this exposure (Akter et al., 2019;Gutyj et al., 2019;Ali et al., 2021;Kar & Patra, 2021).This occurs even on a small scale, as this contaminant is highly absorbed by animals and can cause economic losses (Chen et al., 2021).
Therefore, it is concluded that the use of golden mussels as a source of Ca contaminated with up to 30 mg kg -¹ of Cd does not affect broiler chicken growth performance.However, Cd concentrations above 6.94 mg kg -¹ in broiler chicken diets cause high Cd concentrations in meat and organs, which are above the levels permitted for human consumption.
where: IF = AIA from diet/AIA from excreta.

Table 2
Growth performance of broiler chickens fed with the inclusion of golden mussel shells flour (GMSF) contaminated with increasing inclusions of Cd

Table 3
Cadmium concentrations in different tissues of broiler chickens fed gold mussel shell flour contaminated with increasing inclusions of Cd 1 SEM (Standard error of mean)Singh et al.

Table 5
Serum concentrations of Ca and P of broiler chickens fed with golden mussel shell flour contaminated with increasing inclusion rates of Cd.

Table 6
Deformation, resistance, and flexibility of bones in broilers fed diets containing golden mussel shell flour contaminated with increasing inclusions of Cd

Table 7
Apparent metabolizable Cd (AM Cd), coefficient of apparent digestible Cd (CAD Cd) in broiler chickens fed with golden mussel shell flour contaminated with Cd