Putative cardiotoxic compounds extracted from meat meal as a potential risk factor for the development of heart failure in fast-growing commercial broilers

Abstract

Thermal processing of meat products generates cardiotoxic compounds capable of inducing heart failure in both humans and laboratory animals. Such compounds may be present in broiler diets because supplements such as meat meal (MM), which are commonly used in broiler rations, are rendered at high temperature. Our objective was to evaluate whether putative cardiotoxic compounds in MM increase the risk of heart failure in broilers. The treatment and control diets were prepared by mixing the condensed MM extract (equivalent to dietary MM inclusion of 25%) or placebo (condensed extraction medium) with commercial broiler feed, and the respective diets were offered to commercial male broilers randomly allocated to either treatment or control groups. Broilers fed a diet spiked with MM extract showed a higher incidence (P<0.05) of chronic heart failure (65.5%) in comparison with the control group (55.4%). Postmortem examination upon termination of the experiment revealed that, in comparison with control broilers, broilers fed diet containing MM extract showed higher incidence of lesions indicative of subclinical heart disease evidenced grossly by ventricular dilation and pericardial effusions, microscopically by changes characteristic of cardiomyocyte degeneration, and ultrastructurally by changes in contractile elements and in mitochondria. Measurements of cardiac high-energy phosphates revealed that broilers fed the diet containing MM extract had lower (P<0.05) levels of cardiac energy reserve as compared with birds fed control diet. We conclude that cardiotoxic factors that can induce patho-physiological changes in the heart are present in MM.

Introduction

Selection for traits of economic value has resulted in a broiler genotype that has inherent predisposition to heart failure (Navarro et al., 2006; Druyan et al., 2007). Clinical observations and necropsy findings from several studies indicate that many commercial broilers show subclinical signs of heart condition, and are at higher risk of heart failure (Odom et al., 1992; Owen et al., 1995; Olkowski et al., 1997, 1998, 1999, 2001, 2003; Olkowski, 2007). However, factors that may increase the risk of heart failure in broilers are poorly understood.

Heart failure in laboratory animals and humans has been found to be associated with cardiotoxic compounds generated during thermal meat processing (Davis et al., 1994; Gaubatz, 1997; Dubuisson et al., 2001). Animal by-products such as meat meal (MM) and fish meal (FM) used as dietary supplements in poultry are processed at high temperature during the rendering process, so there is little doubt that cardiotoxic compounds known to be generated during thermal processing of meat (Davis et al., 1994; Gaubatz, 1997; Dubuisson et al., 2001) are present in the MM or FM. Also, some plant protein supplements (e.g. soy bean meal) commonly used in broiler diets are subjected to heat treatment to neutralize anti-nutritional factors, and thus similar compounds may be derived from plant protein supplements (Yoshida et al., 1978; Lan et al., 2004).

Supplements of animal origin such as MM and FM that are commonly used in broiler diets may contain factors that are detrimental to cardiac health. Given the fact that many fast-growing broilers are already predisposed to heart diseases (Navarro et al., 2006; Druyan et al., 2007; Olkowski, 2007), it is possible that any cardiotoxic compounds that may be present in the broiler diet can further increase the risk of heart failure in susceptible individuals. In order to test this hypothesis, the present study was designed to examine whether MM-derived factors contribute to the risk of acute or chronic heart problems in fast-growing broilers. First, we investigated whether acidified methanol extractable factors from MM in the diet increase the incidence of acute or chronic heart failure. We also investigated the effect of these factors on the basic parameters of heart performance, myocardial susceptibility to arrhythmia, and energy metabolism in cardiomyocytes.

Materials and Methods

General

Environmental details, management and feeding regimes were as described previously (Olkowski et al., 1999). Briefly, the birds were housed from 1 day old in an environmentally controlled room under constant light. During the first 7 days the temperature was maintained at 34°C, followed by a gradual decrease to a level approximately 21°C by the end of the third week, and to 17°C by the end of the fifth week. The lowered environmental temperature forces the birds to increase their metabolic rate, which results in increased burden on the cardiovascular system. This approach is very effective in precipitating heart failure practically in all broilers predisposed to heart condition (Nain et al., 2008).

Two experiments were conducted using commercial male broilers randomly allocated to two experimental groups. Each group was replicated twice (Experiment 1) and three times (Experiment 2) using 158-day-old and 238-day-old commercial broilers, respectively. The basic experimental unit was comprised of a group of 35-day-old to 40-day-old male broiler chickens. On day 7, the chickens in the respective control and treatment groups were offered either commercial broiler diet containing placebo (condensed extraction medium) or commercial broiler diet mixed with condensed MM extract. Feed and water were provided ad libitum.

The experimental protocols were approved by the University of Saskatchewan Animal Care Committee and the procedures were performed in accordance with the requirements of the Guide to the Care and Use of Experimental Animals (Canadian Council on Animal Care, 1993).

Meat meal extraction procedure and preparation of diets

The heat-generated cardiotoxic factors were extracted using methods as described by Toribio et al. (2000) with modifications. Commercial MM was extracted with methanol acidified with 1% HCl (90:10 v/v) at a ratio of 1:4 (wt/vol). First, MM was soaked, and then stirred with acidified methanol for 40 min. The extracted liquid was then filtered, and the filtrate was condensed using a rotary evaporator at 45^oC.

The treatment and control diets were prepared by thoroughly mixing the condensed MM extract or placebo (condensed extraction medium) with commercial broiler feed. These preparations were spread thin on trays, and were air-dried overnight. The physical characteristic of the feed was not affected by the process of diet preparation. The MM extract added to treatment diet was equivalent to 25% of MM (wt/wt) of the basal diet (i.e. the evaporated slurry extracted from 1 kg MM was mixed with 4 kg feed).

Clinical monitoring

Birds were monitored several times daily for clinical signs of overt heart disease (fatigue, tachypnea, cyanosis, and ascites and exercise intolerance when stimulated to move, manifested by need to rest, and /or increased respiratory rate and cyanosis after a taking just a few steps.). The physical examination was performed prior to electrocardiographic (ECG) measurements and blood or tissue procurement for biochemical analyses, as well as on birds that were euthanized.

Electrocardiographic measurements

The ECG measurements were obtained from needle electrodes implanted subcutaneously using a lead II arrangement after induction of light anaesthesia as described previously (Olkowski et al., 1997). The signals from the ECG monitor were digitized using an analogue to digital data recording unit and software (Mac Lab and Scope 3.3; AD Instruments Pty Ltd, Castle Hill, Australia) and were processed using a Macintosh computer. Electrocardiographic records were collected from 43 and 31 broilers that appeared normal during routine clinical examination from the control and treatment groups, respectively, during the fifth week of age. The ECG data were evaluated for abnormal heart electrophysiological patterns and QRS axis deviation as described previously (Olkowski et al., 1997).

Blood gas measurements

Blood gas measurements were obtained from 10 randomly selected birds from each group at the end of the fifth week of the experiment, and 10 broilers that developed congestive heart failure (CHF) fed with either diet containing MM extract or placebo were sampled. For blood gas measurements, approximately 0.5 ml blood samples were obtained anaerobically from the wing vein. The samples were analysed for pH, pCO₂, pO₂, and haemoglobin oxygen saturation using a pH/Blood Gas Analyzer (Bayer Corporation, East Walpole, Massachusetts, USA). Physiological values for blood gas parameters as well as values for baseline hypoxemia were established on the basis of several experiments conducted in our lab. Initially we evaluated blood gas values from slow-growing, feed-restricted broilers (low incidence heart failure and hypoxaemia), from fast-growing broilers (high incidence of heart failure and hypoxaemia), and from broilers with advanced heart failure and ascites. As a physiological reference group we used Leghorn chickens (resistant to hypoxaemia and heart disease). Our studies showed that blood gas values in slow-growing broilers are comparable with values found in Leghorn chickens; therefore we assumed these parameters as normal, with venous blood mean values for pO₂ of 42.8 mmHg, and for HbO₂Sat of 78.3% (for details see Olkowski et al., 2005).

Heart tissue procurement for biochemical analysis

At the end of the sixth week of the experiment, heart tissue samples for biochemical analysis (high-energy phosphates and l-carnitine) were obtained from five randomly selected apparently normal birds from each group, and from broilers with clinical signs of CHF. The classification “apparently normal broilers” is based on overt clinical examination, where the subjects do not show typical early clinical signs of CHF such as fatigue, exercise intolerance, tachypnea, cyanosis, or ascites. The hearts were collected immediately following cervical dislocation, snap-frozen in liquid nitrogen, and stored as such until analysed.

Postmortem examination

Detailed gross postmortem examination was performed on all mortalities and birds euthanized during the course of the study. Diagnosis of sudden death syndrome (SDS) was made when death occurred in well-grown, apparently normal birds, without any other cause of death evident upon post mortem examination. The diagnosis of CHF was based on findings of gross dilation of the ventricular chambers along with accumulation of ascitic fluid in the abdominal cavity. At the termination of the experiment, all surviving birds were subjected to gross postmortem examination. The broilers that did not develop fulminant CHF during the course of study but at postmortem examination had dilation of ventricular chambers and pericardial effusions were classified as subclinical cases. These subclinical cases were evaluated for the presence of cardiac lesions such as dilation of ventricular chambers and the amount of pericardial effusions. The dilations of ventricular chambers (Figure 1) were graded as described previously in detail (Olkowski et al., 1998). The pericardial effusions were graded based on the amount of fluid in pericardial sac. A large volume (>5 ml) of pericardial effusion with severely distended pericardium was classified as severe lesion, and was considered a variable indicative of impending heart pump failure (Olkowski et al., 2003).

Figure 1.  Serial sections of broiler heart representing morphological changes characterized as (1a) mild ventricular dilation, (1b) moderate ventricular dilation, and (1c) severe ventricular dilation. RV, right ventricle; LV, left ventricle. It is noteworthy that as the dilation of the ventricular chambers increase, the thickness of ventricular myocardium decreases, which is indicative of degeneration of cardiac tissue.

Measurements of cardiac high-energy phosphates and l-carnitine

Cardiac creatine phosphate (CrP), adenine triphosphate (ATP), adenine diphosphate (ADP), and adenine monophosphate (AMP) were measured in five randomly selected birds from each group, and in five broilers with CHF as previously described (Olkowski et al., 2007). l-Carnitine was measured as described by Feng et al. (2006) with minor modifications. Briefly, samples of myocardium were obtained from the left ventricular free wall taken midway between the apex and atrio-ventricular septum, and were homogenized with ice-cold phosphate buffer (50 mM, pH 7.4) at a ratio of 200 mg tissue/1 ml buffer. The homogenates were centrifuged at 2500×g for 10 min at 4°C. The supernatant was precipitated using acetonitrile and methanol (9:1 v/v). A 300 mg mixture of Na₂HPO₄ and Ag₂O (9:1 wt/wt) and 300 mg KH₂PO₄ were added. This preparation was vortexed for 1 h, and following this a derivatizing reagent (40 mg/ml p-bromophenacyl bromide with 50 µl of 40% tetrabutylammonium hydroxide) was added into the organic extract. The reaction mixture was incubated at 60°C for 2 h followed by centrifugation at 12 000×g for 15 min, and the supernatant was used for analysis. l-Carnitine was resolved using a high-performance liquid chromatograhpy system (Agilent 1050) with a Hyperclone 5 µ CN column (Phenomenex, USA). The mobile phase (90% acetonitrile/10 mM citric-phosphate buffer, adjusted to pH 3) was delivered at a flow rate of 1 ml/min. The elution of carnitine was monitored at 260 nm.

Light and transmission electron microscopy

The heart from three birds fed the control and treatment diets were processed for microscopic examination immediately after cervical dislocation at the termination of experiment (6 weeks of age). The hearts used for histopathology were visually assessed for lesions and did not show any lesions upon necropsy. For light microscopy, samples of heart tissue taken were obtained from the left ventricular free wall taken midway between the apex and atrio-ventricular septum, were fixed in formaldehyde buffer and, following fixation, blocks of myocardium were embedded in paraffin. Several longitudinal and transverse sections (5 µm) were processed for light microscopy and were stained with haematoxylin and eosin. For electron microscopy, samples of heart tissue were fixed in glutaraldehyde, and were further processed as described previously by Olkowski et al. (2001). Multiple ultrathin sections were obtained using microtome on copper grids and were stained with uranyl acetate and lead citrate. Electron micrographs were taken on a Philips 410 LS transmission electron microscope.

Statistical analysis

Data were analysed using the microcomputer package Number Cruncher Statistical System (Hintze, 1995). Energy parameters, l-carnitine and blood gas data were analysed using analysis of variance and the means were separated by using Fisher's least significant difference test (LSD). The incidence of CHF, SDS and ECG data were analysed using Fisher's exact test. Morbidity/mortality and descriptive clinical data were analysed using data combined from Experiments 1 and 2. Statistical significance was assumed to exist when the probability of making a type I error was less than 0.05.

Results

Clinical and pathological observations

All broilers appeared normal on overt clinical examination at the commencement of the treatment (day 7). Feed consumption was not affected by the dietary treatment.

Overall, the data from both experiments showed consistently that broilers fed diet containing MM extract were at higher risk (P < 0.05) of succumbing to fulminant CHF (65.4%) than those fed the placebo containing diet (55.4%) (for details, see Table 1). The incidence of acute heart failure (SDS) was not significantly different (P > 0.05) between the groups fed the diet containing MM extract or the diet with placebo.

Table 1.  Incidence of CHF and SDS in broilers fed the diet spiked with MM extract (TRT) and those fed the placebo diet (CTR)

	Experimental group	CHF (mortality/morbidity)	SDS (mortality)
Experiment 1	CTR (n=83)	38/83^A (45.8%)	5/83^A (6.0%)
	TRT (n=75)	39/75^A (52.0%)	6/75^A (8.0%)
Exepriment 2	CTR (n=119)	74/119^B (62.2%)	7/119^A (5.9%)
	TRT (n=119)	88/119^A (73.9%)	8/119^A (6.7%)
Experiments 1 and 2 combined data	CTR (n=202)	112/202^B (55.4%)	12/202^A (5.9%)
	TRT (n=194)	127/194^A (65.5%)	14/194^A (7.2%)
Significance (P value, Fisher's exact test)		<0.05	0.38
Data presented as the frequency of occurrence (% of birds examined). n, number of birds. Frequencies of occurrence within each column in each experiment with different uppercase superscript letters are significantly different (P<0.05).

All birds that developed CHF (data combined from both treatments) showed severe hypercapnia (pCO₂=55.0 mmHg), hypoxaemia (pO₂=21.1 mmHg) and the lowest haemoglobin oxygen saturation (HbO₂Sat = 31.6%) as compared with birds fed the diet containing MM extract or the diet with placebo (Table 2). The blood gas measurements in apparently normal randomly derived broilers were indicative of hypoxaemia in both groups, with values for pO₂ (39.4 mmHg) and HbO₂Sat (72.0%) in the group fed the diet containing MM extract being slightly lower than in broilers fed the diet with placebo (pO₂=41.5 mmHg and HbO₂Sat = 77.1%), but the differences were not significant.

Table 2.  Comparative study of CrP, ATP, ADP, AMP and l-carnitine content in the left ventricular myocardium in apparently normal broilers fed with placebo diet (CTR), in broilers fed the diet spiked with MM extract (TRT) and in broilers with CHF

Parameters	CTR	TRT	Broiler with CHF	Analysis of variance P value
pH (units)	7.42±0.008^B	7.39±0.021^AB	7.34±0.019^A	<0.001
pCO2 (mmHg)	45.1±1.32^A	45.6±1.42^A	55.0±4.13^B	<0.01
pO2 (mmHg)	41.5±1.54^B	39.4±2.02^B	21.1±2.12^A	<0.01
HbO2Sat (%)	77.1±1.76^B	72.0±3.65^B	31.6±6.07^A	<0.01
CrP (µg/mg heart tissue)	1.06±0.020^B	0.81±0.025^A	0.76±0.014^A	<0.001
ATP (µg/mg heart tissue)	1.52±0.045^B	1.35±0.108^B	1.06±0.096^A	<0.01
ADP (µg/mg heart tissue)	0.86±0.017^B	0.66±0.052^B	0.731±0.027^AB	<0.05
AMP (µg/mg heart tissue)	0.28±0.035	0.33±0.034	0.37±0.052	0.38
l-Carnitine (µM/mg heart tissue)	0.30±0.033	0.24±0.070	0.16±0.013	0.16
Data presented as the mean±standard error, Means within rows with different uppercase superscript letters are significantly different (P < 0.05).

Postmortem examination in all birds showing signs of CHF revealed gross dilation of the ventricles and severe pericardial effusions along with ascitic fluid in abdominal cavity. There were no differences in gross features of heart pathology between CHF broilers from the placebo group and the CHF broilers fed diets spiked with MM extract. However, among apparently normal birds from both groups that showed pathological changes indicative of subclinical heart disease, the occurrence of severe ventricular dilation was higher (P < 0.05) in the group fed the diet containing MM extract (59.5% of examined), as compared with the placebo group (40.7% of examined). The presence of severe pericardial effusions (>5 ml) was found in 44.6% of apparently normal broilers exposed to MM extract, compared with 27.8% of broilers fed the diet with placebo (P = 0.06).

Electrocardiographic measurements

Electrocardiographic evaluation revealed that 6.97% of broilers fed the control diet, and 12.9% of broilers fed the treated diet showed arrhythmia. Cardiac rhythm abnormalities included atrio-ventricular blocks, and episodes of atrial and ventricular arrhythmia, but, most commonly, premature ventricular contractions were observed. Analysis of QRS axis deviation revealed that 44.2% of broilers fed the placebo diet and 51.6% of broilers fed the diet spiked with MM extract showed left axis deviation, an early sign of CHF. The heart rate in broilers that developed CHF (235±5.7 beats/min) was significantly lower (P < 0.001) than the heart rate in apparently normal broilers either from the group fed the placebo-containing diet (308±6.4 beats/min) or the group fed the MM-spiked diet (310±7.0 beats/min).

Light microscopic and ultrastructural changes

Histopathological examination revealed degenerative changes in the myocardium of apparently normal broilers from both groups, but there were considerable differences in the magnitude of the lesions (Figure 2). Ultrastructural examination revealed morphological changes in the mitochondrial architecture, characterized by swelling, vacuolization and destruction of matrices and cristae (Figure 3). Overall, cardiomyocytes from broilers fed the placebo containing diet showed mostly normal morphology of mitochondria, but mild to moderate changes were apparent in some clusters of mitochondria (Figure 3a). In contrast, cardiomyocytes of broilers fed the diet containing MM extract contained numerous mitochondria showing more advanced morphological changes (Figure 3b).

Figure 2.  2a: Generic histo-pathological features of the mural left ventricular myocardium in a broiler indicative of a subclinical heart condition. Representative micrographs showing the extent of the lesions in (2b) broilers from the group fed the placebo diet and (2c) broilers fed the diet spiked with MM extract. Bars: 2a = 25 µm, 2b and 2c = 50 µm. In broilers with a subclinical heart condition (2a), typical lesions in the ventricular myocardium consisted of degenerative changes in the cardiomyocytes characterized by distinct dull darker pink appearance of affected cardiomyocytes with cytoplasmic eosinophilia and signs of chromatine condensation (white arrows). More advanced stages were evidenced by nuclear pyknosis and karyorrhexis (black arrows). Some cardiomyocytes showed vacuolated cytoplasm and degenerative changes in nuclei indicative of advanced changes (blue arrows). The lesions appear to have similar qualitative features in both groups, but in comparison with birds from the placebo group (2b), those fed the diet containing MM extract (2c) showed more extensive degenerative changes in the ventricular myocardium.

Figure 3.  Representative transmission electron micrographs from the left myocardium of (3a) a broiler fed the placebo-containing diet and (3b) a broiler fed the diet containing MM extract. Bar = 1 µm. For the most part, myocardial mitochondria showed normal morphology with well-defined and dense cristae, but in some areas of the cardiomyocytes degenerative changes in the mitochondrial structure were evident. Examples of typical changes in mitochondrial morphology in broilers fed the placebo-containing diet (3a) and broilers fed the diet spiked with MM extract (3b). It is noteworthy that the changes in the mitochondria (arrows) such as swelling, vacuolization, and destruction of matrices and cristae were qualitatively evident in both groups, but are more severe in broilers fed the diet spiked with MM extract in comparison with broilers fed the placebo-containing diet.

Biochemical changes

Cardiac levels of ATP and CrP were significantly (P < 0.01) lower in broilers with CHF (Table 2).

The levels of cardiac ATP and CrP were approximately 16% and 24% lower in birds fed the diet spiked with MM extract as compared with birds fed the placebo diet, but this difference was statistically significant (P < 0.05) only for CrP. The ADP level was lower (P < 0.05) in birds fed the diet spiked with MM extract, as compared with birds fed the placebo diet. The cardiac AMP level was higher by 17.1% and the l-carnitine level was lower by 18.9% in birds fed the diet containing MM extract than in those fed the placebo diet.

Discussion

The findings from the present study indicate that methanol extracted factors from meat meal significantly increase the risk of CHF in fast-growing broilers. The nature and severity of changes in the heart muscle seen on gross examination, as well as by light microscopy and electron microscopy on the three birds examined per group, were consistently more pronounced in birds fed the diet containing MM extract as compared with birds fed the placebo diet. Taken together, these findings indicate that MM used in the present study may contain quantities of compounds capable of inducing cardiac muscle lesions and may precipitate heart failure. Although the identification of specific components of our extract was beyond the scope of present study, it is reasonable to assume that the adverse cardiac effects observed in the present study were associated with several putative cardiotoxic compounds in the MM extract.

Among many compounds that may be produced during high-temperature meat processing, the most recognized are cardiotoxic heterocyclic amines (HA). Synthesis of HA during thermal treatment of proteins is well documented in the literature (Davis et al., 1994; Gaubatz, 1997; Dubuisson et al., 2001; Bordas et al., 2004). Thus far, 20 HA generated in cooked meat have been identified (Bordas et al., 2004). The amounts and types of HA formed during cooking can be attributed to parameters such as time and temperature (Gross et al., 1993; Knize et al., 1998), and in these terms the processing technology used in rendering process of animal byproducts or thermal treatment of plant proteins provide ideal conditions for generation of cardiotoxic HA. Therefore, given the fact that production of MM involves long-term, high-temperature treatment during the rendering process, it is reasonable to assume that these compounds are present in MM used for broiler diets, resulting in increased risk of CHF in susceptible birds.

The histopathological changes observed in the cardiomyocytes of broilers exposed to MM extract are consistent with lesions associated with cardiotoxic effects of HA in experimental animals, such as myocyte necrosis and degeneration, myofibrillar loss, swelling and vacuolization of mitochondria described by Davis et al. (1994). It is known that a free radical mechanism is involved in HA-induced mitochondrial abnormality (Davis & Snyderwine, 1995). The lowered energy metabolism in birds fed MM extract may be a consequence of severe mitochondrial damage in response to increased production of free radicals. In particular, morphological changes in the mitochondria seen in our broilers are remarkably similar to the lesions observed in the mitochondria of monkeys treated with purified 2-amino-3-methylimidazo quinoline (Thorgeirsson et al., 1994), and in rats treated with 2-amino-1-methyl-6-phenylimidazo pyridine (Takahashi et al., 1996). Of note, these compounds are major HA produced during thermal processing of meat (Gross et al., 1993; Guy et al., 2000; Turesky et al., 2005).

The morphological changes in the mitochondria are consistent with the biochemical dysfunction in cardiac energy metabolism. Interestingly, closer scrutiny of the biochemical data from apparently normal broilers fed the placebo diet, those fed diets spiked with MM extract, and broilers with CHF revealed an apparent trend that deserves further consideration. In order to understand the trends in energy parameters in these three groups of birds, the measurements from birds fed the diet containing MM extract and birds with CHF were expressed with reference to levels in broilers fed the placebo diet taken as 100%. These comparisons are presented in Figure 4.

Figure 4.  Relative changes in cardiac CrP, ATP, ADP, AMP and l-Carnitine contents in broilers fed the diet spiked with MM extract (TRT), and in broilers with congestive heart failure (Broiler-CHF) as compared with broilers fed the placebo diet (CTR). Relative values for broilers fed the diet spiked with MM extract (TRT), and for Broiler-CHF are expressed on a percentage basis of data from CTR broilers representing on the graph a reference value of 100.

Insufficiency of energy substrate has recently been linked with this deterioration of heart function in broilers (Olkowski et al., 2007; Nain et al., 2008). The loss of contractile function of the heart is associated with mitochondrial inability to supply ATP and to replenish CrP (energy reservoir) from creatine in the myocardium, leading to a state of energy deprivation in the heart (Stanley et al., 2005).

The lower status of the biochemical parameters critical in the cardiac energy metabolism such as CrP, ATP, and l-carnitine in broilers fed the diet spiked with MM extract relative to the broilers fed the placebo diet may be subsequent to morphological changes seen in the mitochondria. Given the fact that the pattern of changes in all of the biochemical parameters examined in the present study showed a similar trend in both broilers fed diets containing MM extract and in broilers that developed CHF, it is reasonable to assume that biochemical changes associated with the putative cardiotoxic compounds present in MM play a significant role in the development of heart failure.

The present study provides evidence that MM used in commercial broiler diets may contain compounds capable of inducing morphological and biochemical changes in the heart tissue. The use of MM in animal feeds is completely banned in some countries, but in most jurisdictions the ban is restricted only to ruminant animals feed. In North America, MM is commonly used in diet formulation for mono-gastric species, and in particular is widely used in poultry feeds. However, in the feed industry it is a well-known fact that the quality of these products (particularly MM) is highly variable. Undoubtedly, depending on the animal by-products used, and the different rendering technologies, there is possibility that some MM may contain high levels of cardiotoxic compounds. Since relative to other protein supplements MM is frequently competitively priced, there are tendencies to increase use of MM in broiler diet formulation. In the context of our findings, further studies need to be conducted to evaluate the risks associated with various MM and FM products, to establish safe maximum levels of inclusion and to develop rendering processing guidelines and quality control.

Acknowledgements

The research presented in this paper was supported by grants provided by the Poultry Industry Council of Canada and the Natural Sciences and Engineering Research Council of Canada. The authors would like to acknowledge Bingbing Ling for providing technical support in analysing l-carnitine.