Natural and eco-friendly approaches to control aflatoxins in poultry feed

Final Report for GNE15-113

Project Type: Graduate Student
Funds awarded in 2015: $14,393.00
Projected End Date: 12/31/2016
Grant Recipient: University of Connecticut
Region: Northeast
State: Connecticut
Graduate Student:
Faculty Advisor:
Michael Darre
University of Connecticut
Faculty Advisor:
Dr. Kumar Venkitanarayanan
University of Connecticut
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Project Information

Summary:

Aflatoxins (AF) are toxic metabolites primarily produced by molds, Aspergillus flavus and Aspergillus parasiticus. Contamination of poultry feed with AF is a major concern to the poultry industry since aflatoxicosis in chickens results in significant economic losses due to poor feed utilization, decreased body weight, and increased mortality. The overall objective of this proposal is to investigate the efficacy of two food-grade phytochemicals, namely carvacrol (CR) and trans-cinnamaldehyde (TC), in decreasing AF production by Aspergillus spp. in chicken feed and controlling aflatoxicosis in chickens. In the first study, we investigated the inhibitory effect of CR and TC on Aspergillus flavus and Aspergillus parasiticus growth and AF production during long-term storage in chicken feed. Two hundred gram portions of chicken feed supplemented with CR and TC (0%, 0.4%, 0.8%, and 1.0%) were inoculated with A. flavus (NRRL 3357) or A. parasiticus (NRRL 4123) and stored at 25oC for 3 months. Carvacrol and TC significantly inhibited A. flavus and A. parasiticus growth and AF production in chicken feed during the entire storage period (P < 0.05). All the concentrations of CR and TC decreased AF concentrations in the feed to levels below the FDA regulated limit (20 ppb). However, feed samples with no added CR or TC yielded more than 30 ppb (NRRL 4123 and NRRL 3357) of AF. The second study was conducted to evaluate the efficacy of CR and TC in controlling aflatoxicosis in chickens. A total of 240 chickens were fed with AF contaminated feed (~2.5 µg/g) with or without supplementation of 0.75% CR or TC for 5 weeks. In weeks 2, 3, 4, and 5, chicken performance traits, including body weight, feed intake, and feed conversion rate were measured. In addition, the relative weights of liver, spleen, and bursa of Fabricius of birds were determined, and histological analysis of liver was performed. Results revealed that CR and TC supplementation in AF-contaminated feed ameliorated AF-induced adverse effects in chickens. In addition, phytochemical supplementation significantly decreased relative liver weight and improved relative bursa of Fabricius weight in birds, as compared to AF-treated group (P < 0.05). Histological analysis revealed that CR and TC reduced AF-induced toxic effects in the liver of birds, where phytochemical-treated chickens had decreased hepatocellular degeneration, necrosis and inflammation in the liver as compared to chickens fed with AF alone. In the third study, we determined the heat stability of CR and TC in chicken feed. Briefly, chicken feed mixed with 0.75% CR or TC was heated at 150oF or 180oF and stored at room temperature for 2 months. At the 0, 1, and 2 months, CR and TC concentrations in the feed of each treatment were quantified using Gas Chromatography-Mass Spectrometry (GC-MS) analysis. Results revealed that CR and TC maintained high heat stability when treated at 150oF or 180oF, and the two temperatures did not significantly affect the concentrations of CR and TC in the chicken feed for up to 2 months as compared to the untreated control (P > 0.05). Results supported our hypothesis that CR and TC supplementation can attenuate AF-induced adverse effects in chickens.

Introduction:

Aflatoxins (AF) are a group of fungal toxic metabolites of Aspergillus flavus and Aspergillus parasiticus, which can frequently contaminate a variety of feed ingredients, including peanuts, corn and cottonseed (Oguz et al., 2000; Sur and Celik, 2003). Aflatoxins possess potent toxicological and hepatocarcinogenic properties to both animals and humans, and Aflatoxin-B1 has been listed as group I human carcinogen by the International Agency for Research on Cancer (Yunus et al., 2011).

Chickens are primarily exposed to AF through ingestion of contaminated feed. Contamination of poultry feed with AF is a major concern in the poultry industry due to significant economic losses associated with poor feed utilization, decreased body weight gain, and increased mortality (Qureshi et al., 1998; Tessari et al., 2006, Oguz, 2011). Once AF are ingested by chickens, they accumulate in various parts of birds, especially in liver, kidney, and spleen, causing hemorrhagic syndrome, fatty liver syndrome, renal tissue malfunction, and increasing susceptibility to other infections. Moreover, AF residues can be found in several edible tissues of chickens, particularly in chicken meat and egg (Jacobson and Wiseman, 1974; Sudhakar, 1992; Qureshi et al, 1998), thereby constituting a potential threat to consumer health.

The Food and Agriculture Organization reported that 25% of the world’s grains are contaminated by mycotoxins, and AF contamination is the most common among them. The economic losses due to AF contamination to the US poultry industry exceed $143 million annually (CAST, 1989). Currently cost-effective and practical methods to detoxify aflatoxin contamination on a large scale are limited. At present, the inclusion of AF-binding adsorbent in feeds is employed to protect animals against the harmful effects of AF. However, several adsorbents have been shown to impair nutrient utilization (Kubena et al., 1993) and mineral absorption in animals (Edrington et al., 1997). It was concluded that none of the current strategies of controlling AF is sufficient to completely fulfill the necessary safety and cost requirement (Teniola et al., 2005), thereby highlighting the need for an effective strategy to control AF in feeds.  

Phytochemicals are a group of natural, plant-derived compounds that have traditionally been used as food preservatives and flavor enhancers. In the past decade, the use of phytochemicals as effective antimicrobials has gained significant attention due to their non-toxic nature, and increasing concern over the safety of synthetic chemicals and emerging antibiotic-resistant strains of microorganisms (Salamci et al., 2007). Among the various phytochemicals, carvacrol (CR), listed as generally recognized as safe (GRAS) by the FDA, is a major component in oregano oil obtained from Origanum vulgare (Lamiaceae). Carvacrol has been found effective against bacterial and fungal infections of the gastrointestinal and genitourinary tract (Adams et al., 2004; Chun et al., 2005) as well as against a wide range of pathogens, including Staphylococcus aureus, Escherichia coli, and Streptococcus pneumoniae (Hersch-Martinez et al., 2005). Trans-cinnamaldehyde (TC) is another GRAS-status ingredient present in the bark extract of cinnamon (Cinnamomum zeylandicum). Various studies have demonstrated the antimicrobial properties of TC against both gram-negative and -positive bacteria (Burt, 2004; Upadhyaya et al., 2014).

Our preliminary research pertaining to this proposal showed that CR (0.04 and 0.08%) and TC (0.01 and 0.02%) significantly reduced A. flavus and A. parasiticus growth in potato dextrose broth. Moreover, 0.02% CR and 0.005% TC inhibited AF production by A. flavus and A. parasiticus by 95% and 75% when compared to the control, respectively. In addition, the effective concentrations of CR and TC for inhibiting AF production by molds in chicken feed were identified, where CR and TC at 0.8 and 1.0% significantly inhibited A. flavus and A. parasiticus growth and AF production in chicken feed on days 5 and 7 of storage. Based on these preliminary results, we hypothesized that CR and TC could be effective in reducing AF contamination in poultry feed during long-term storage and attenuating aflatoxin-mediated pathogenesis in chickens.

References:

Adams, T. B., S. M. Cohen, J. Doull, V. J. Feron, J. I. Goodman, L. J. Marnett, P. S. Portoghese, R. L. Smith, W. J. Waddell, and B. M. Wagner. (2004) The FEMA GRAS assessment of cinnamyl derivatives used as flavor ingredients. Food Chem Toxicol 42.2:157-185.

Burt, S. (2004) Essential oils: their antibacterial properties and potential application in foods: a review. Int J Food Microbiol 94:223-253.

CAST. (1989) Mycotoxins: economic and health risks. Council for Agricultural Science and Technology, 137 Lynn Avenue, Ames, IA 50010.

Chun, S. S., D. A. Vattem, Y. T. Lin, and K. Shetty. (2005) Phenolic antioxidants from clonal oregano (Origanum vulgare) with antimicrobial activity against Helicobacter pylori. Pro Biochem 40:809-816.

Edrington, T. S., L. F. Kubena, R. B. Harvey, and G. E. Rottinghaus. (1997) Influence of a superactivated charcoal on the toxic effects of aflatoxin or T-2 toxin in growing broilers. Poult. Sci. 76:1205-1211.

Hersch-Martinez, P., B. E. Leanos-Miranda, and F. Solorzano-Santos. (2005) Antibacterial effects of commercial essential oils over locally prevalent pathogenic strains in Mexico. Fitoterapia 76:453-457.

Jacobson, W. C., and H. G. Wiseman. (1974) The transmission of aflatoxin B1 into eggs. Poult Sci 53:1743-1745.

Kubena, L. F., R. B. Harvey, W. E. Huff, M. H. Elissalde, A. G. Yersin, T. D. Phillips, and G. E.  Rottinghaus. (1993) Efficacy of a hydrated sodium calcium aluminosilicate to reduce the toxicity of aflatoxin and diacetoxyscirpenol. Poult. Sci. 72:51-59.

Oguz, H., T. Kececi, Y. O. Birdane, F. Onder, and V. Kurtoglu. (2000) Effect of clinoptilolite on serum biochemical and haematological characters of broiler chickens during aflatoxicosis. Res in Vet Sci 69:89-93.

Oguz, H. (2011) A review from experimental trials on detoxification of aflatoxin in poultry feed. Eurasian J Vet Sci 27:1-12.

Qureshi, M. A., J. Brake, P. B. Hamilton, W. M. Hagler, and S. Nesheim. (1998) Dietary exposure of breeders to aflatoxin results in immune dysfunction in progeny chicks. Poult Sci 77:812-819.

Salamci, E., S. Kordali, R. Kotan, A. Cakir, and Y. Kaya. (2007) Chemical compositions, antimicrobial and herbicidal effects of essential oils isolated from Turkish Tanacetum aucheranum and Tanacetum chiliophyllum var. chiliophyllum. Biochem. Syst. Ecol.35:569-581.

Sudhakar, B. V. (1992) The Carry-over effect of aflatoxin-B1 into eggs and liver of chickens. Indian Vet J 69.11:1061-1062.

Sur, E. and I. Celik. (2003) Effects of aflatoxin B1 on the development of the bursa of Fabricius and blood lymphocyte acid phosphatase of the chicken. Br Poult Sci 44:558-566.

Teniola, O. D., P. A. Addo, I. M. Brost, P. Färber, K. D. Jany, J. F. Alberts. (2005) Degradation of aflatoxin B1 by cell-free extracts of Rhodococcus erythropolis and Mycobacterium fluoranthenivorans sp. nov. DSM44556T. Int. J. Food Microbiol. 105:111-117.

Tessari, E. N. C., C. A. F. Oliveira, A. L. S. P. Cardoso, D. R. Ledoux, and G. E. Rottinghaus. (2006) Effects of aflatoxin B1 and fumonisin B1 on body weight, antibody titres and histology of broiler chicks. Br. Poult. Sci. 47:357-364.

Upadhyaya, I., A. Kollanoor-Johny, M. J. Darre, and K. Venkitanarayanan. (2014) Efficacy of plant-derived antimicrobials for reducing egg-borne transmission of Salmonella Enteritidis. J Appl Poul Res 23.2:330-339

Yunus, A. W., E. Razzazi-Fazeli, and J.  Bohm. (2011) Aflatoxin B1 in affecting broiler’s performance, immunity, and gastrointestinal tract: a review of history and contemporary issues. Toxins 3:566-590.

Project Objectives:

The overall objective of this proposal is to improve poultry feed safety, bird health, and poultry food safety by using natural and environment-friendly approaches to control aflatoxins. The specific objectives include: Objective 1. To investigate the efficacy of carvacrol (CR) and trans-cinnamaldehyde (TC) in reducing Aspergillus flavus and A. parasiticus growth and aflatoxin (AF) production in chicken feed during long-term storage. Objective 2. To determine the efficacy of in-feed supplementation of CR and TC in reducing aflatoxicosis in chickens. Objective 3. To study the heat stability of CR and TC treated with commercial manufacturing temperatures in chicken feed.

Cooperators

Click linked name(s) to expand
  • Dr. Michael Darre
  • Dr. Kumar Venkitanarayanan

Research

Materials and methods:

To investigate the efficacy of CR and TC in reducing A. flavus and A. parasiticus growth and AF production in chicken feed during long-term storage, A. flavus NRRL 3357 and A. parasiticus NRRL 4123 obtained from USDA-ARS culture collection were used in this study. Poultry-grower crumble feed procured from the University of Connecticut poultry farm was inoculated with each mold separately (Kusumaningtyas et al, 2006), wherein A. flavus NRRL 3357 or A. parasiticus NRRL 4123 was added to 200 g portions of feed to obtain ~5 log CFU/g of mold count, and mixed well. After inoculation, the feed was added with CR or TC at 0%, 0.4%, 0.8% or 1.0%, and stored in sealed plastic bags at 25oC for 3 months. A twenty-gram portion of the feed was sampled on weeks 0, 1, 2, 3, 4, 8 and 12, of which 10 g was used for mold enumeration and the remaining 10 g for AF quantitation.

        To enumerate A. flavus and A. parasiticus in control and treated feed, 10 g portions of feed samples were added to 40 ml of phosphate buffered saline (PBS, pH 7.0) in sterile whirl-pak bags (Sigma-Aldrich), and pummeled in a stomacher for 1 min. The feed homogenate was serially diluted (1:10) in PBS, and 0.1 ml aliquots from appropriate dilutions were surface plated on duplicate PDA plates, and incubated. The AF in the feed was quantitated using the commercial ELISA kit (Romer Labs).

In the second study, to determine the efficacy of in-feed supplementation of CR and TC in reducing aflatoxicosis in chickens, a total of 240 chickens were randomly assigned to 6 different treatments with 20 birds per pen and two pens per treatment. The 6 treatment groups included: (1) Control (feed with no AF and no CR/TC supplementation), (2) CR control (feed with no AF, but 0.75% supplemental CR), (3) TC control (feed with no AF, but 0.75% supplemental TC), (4) AF (feed containing ~2.5 µg/g AF), (5) AF+CR (feed containing ~2.5 µg/g AF and 0.75% supplemental CR), and (6) AF+TC (feed containing ~2.5 µg/g AF and 0.75% supplemental TC). This concentration was selected based on previous studies from our laboratory, where the results showed that 0.75% TC produced no deleterious effects on chicken performance (Kollanoor-Johny et al., 2012). All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Connecticut.

Five birds per pen per treatment were euthanized using CO2 asphyxiation in weeks 2, 3, 4, and 5. Individual body weight (BW) was measured at each time point. Average feed intake and body weight gain was corrected for mortality when calculating feed conversion ratio (FCR) as kg feed consumed/ kg body weight gain for each treatment. At each time point (week 2, week 3, week 4, and week 5), entire organs of liver, spleen, and bursa of Fabricius were collected and weighed (Aravind et al., 2003). Relative organ weights were calculated as a percentage of BW. In weeks 3 and 5, a portion of liver from three different birds from each group was rapidly fixed in 10% neutral buffered formalin solution for at least 24 hours, and stored at room temperature until subjected to histological analysis (Tedesco et al., 2004). The liver samples were stained with hematoxylin and eosin (H&E), according to the method described by Culling (1983). The slides were examined using light microscope equipped with digital camera.

For the third study, we determined the heat stability of CR and TC treated with commercial manufacturing temperatures in chicken feed. One kilogram of the feed was supplemented with or without 0.75% of CR or TC and subjected to heat treatment at 150oF and 180oF, respectively. The treated and untreated feed was packed and stored at 25oC for 2 months. One hundred gram portions of feed were collected immediately after treatment, and at 1 and 2 months of storage period. The feed samples were analyzed for TC and CR content by Gas Chromatography-Mass Spectrometry (GC-MS) on a Varian 450-GC coupled to a 320-MS triple quad mass spectrometer (Bruker Daltonics, Billerica, MA). Separation was achieved with a Phenomenex Zebron ZB-5HT Inferno column to detect the presence and the concentration of CR and TC in each sample. Triplicate samples of each treatment and control were analyzed in this experiment.

The data from the first study were analyzed using the PROC-MIXED procedure of the statistical analysis software (version 9.1, SAS Institute Inc. Cary, NC). In the second and the third study, the data were analyzed using the PROC-GENMOD procedure of Statistical Analysis Software (SAS, version 9.3, SAS institute, Inc.). Differences among the means were detected at P < 0.05 using Fisher’s Least Significance Difference (LSD) test with appropriate correction for multiple comparisons.

References:

Abdollahi, M. R., V. Ravindran, and B. Svihus, B. (2013). Pelleting of broiler diets: An overview with emphasis on pellet quality and nutritional value. Animal Feed Sci. Technol. 179(1), 1-23.

Aravind, K. L., V. S. Patil, G. Devegowda, B. Umakantha, and S. P. Ganpule. (2003) Efficacy of esterified glucomannan to counteract mycotoxicosis in naturally contaminated feed on performance and serum biochemical and hematological parameters in broilers. Poult Sci 82:571-576.

Culling, C. F. A. (1983) Handbook of histopathological and histochemical techniques: including museum techniques. Butterworth-Heinemann.

Gholivand, M. B. and F. Ahmadi (2008) Simultaneous determination of trans-cinnamaldehyde and benzaldehyde in different real samples by differential pulse polarography and study of heat stability of trans-cinnamaldehyde. Anal. Lett. 41(18), 3324-3341.

Kollanoor-Johny, A., T. Mattson, S. A. Baskaran, M. A. Amalaradjou, S. Babapoor, B. March, S. Valipe, M. Darre, T. Hoagland, D. Schreiber, M. I. Khan, A. Donoghue, D. Donoghue, and K. Venkitanarayanan. (2012) Reduction of Salmonella enterica serovar Enteritidis colonization in 20-day-old broiler chickens by the plant-derived compounds trans-cinnamaldehyde and eugenol. Appl Environ Microbiol 78.8:2981-2987.

Kusumaningtyas, E., R. Widiastuti, and R. Maryam. (2006) Reduction of aflatoxin B1 in chicken feed by using Saccharomyces cerevisiae, Rhizopus oligosporus, and their combination. Mycopathologia. 162:307-311.

Tedesco, D., S. Steidler, S. Galletti, M. Tameni, O. Sonzogni, and L. Ravarotto. (2004) Efficacy of silymarin-phospholipid complex in reducing the toxicity of aflatoxin B1 in broiler chicks. Poult Sci 83:1839-1843.

Research results and discussion:

Results

In our first study, we observed that CR and TC exhibited a significant inhibitory effect on A. flavus and A. parasiticus growth and AF production in poultry feed. The growth of both molds was significantly decreased by 0.8 and 1.0% CR from weeks 4 through 12, with 4.0 log and 3 log CFU/mL reductions in A. flavus and A. parasiticus populations, respectively at the end of the storage period (Fig. 1). However, irrespective of the concentration, CR (0.4%, 0.8%, and 1.0%) decreased AF production by both molds by more than 60% at 12 weeks compared to the control (P < 0.05) (Fig. 1). In addition, TC (0.8% and 1.0%) reduced the counts of both A. flavus and A. parasiticus throughout the storage period in the chicken feed (P < 0.05) and all concentrations of TC inhibited AF production by approximately 60% by the end of storage period (P < 0.05) (Fig. 2).

Our second study investigated the efficacy of in-feed supplementation of CR and TC in reducing aflatoxicosis in chickens. The average BW of each experimental group is shown in Figure 3. In week 4 and 5, control birds had the maximum BW. Aflatoxin supplementation at 2.5 µg/g significantly reduced chicken BW in week 4 and week 5 by ~11% and ~18%, respectively as compared to the control (P < 0.05). Although the results were not significant (P > 0.05), birds supplemented with TC at 0.75% were generally heavier than the AF-treated birds. However, in-feed supplementation of CR at 0.75% significantly increased the BW of the birds fed with 2.5 µg/g AF by ~ 13% as compared to chickens fed with AF alone in week 5. However, feed intake and FCR were not significantly affected by the treatments.

Table 1 shows the effect of CR and TC on the relative liver weight in chickens exposed to AF. Results indicated that the relative liver weight from AF-treated chickens was significantly greater in week 3 and week 5 as compared to the control (P < 0.05). In addition, in-feed supplementation of CR and TC significantly decreased the relative liver weight in birds compared to AF-treated group (P < 0.05). Similarly, chickens supplemented with AF-contaminated feed demonstrated a significantly decreased relative bursa of Fabricius weight compared to other treatment groups (Table 2). No significant differences in relative bursa of Fabricius weight were observed among other treatment groups throughout the study.

For the liver histopathological analysis, liver samples from control, 0.75% CR control, and 0.75% TC control revealed no pathological lesions and appeared normal. Liver samples from AF-supplemented chickens demonstrated moderate to severe hepatocellular degeneration, necrosis, and lymphoplasmacytic infiltration. However, the liver from birds fed with AF + CR or TC showed a lesser inflammatory infiltration and minimal hepatocellular degeneration and necrosis compared to the birds fed with AF alone (Fig. 4). 

In the third study, we investigated the heat stability of CR and TC treated with commercial manufacturing temperatures in chicken feed, where we observed that the recovery rate (%) of CR and TC treated at 150oF and 180oF in the feed was not affected as compared to the untreated controls on day 0 (P > 0.05). A similar trend was seen at the 1 month and 2 month time point, depicting that that heat treatment of feed did not adversely affect the stability of the phytochemicals when compared to untreated feed (P > 0.05). At the end of the 2 month storage, at least 87% of CR and 77% of TC treated at 180oF were still recovered as compared to the control (Fig. 5).

Discussion

In the first study, we investigated the efficacy of CR and TC, two naturally occurring plant compounds in inhibiting A. flavus and A. parasiticus growth and AF production. Although both phytochemicals were effective in reducing AF production by A. flavus and A. parasiticus, no consequential association between the mold growth and AF production was observed (Fig. 1 and Fig. 2). These findings concur with the study by Kusumaningtyas et al. (2006), who reported no correlation between the growth of A. flavus and AF production. Similarly, Bluma and Etchaverry (2006) reported that when A. flavus was grown in maize in the presence of Bacillus strains, the reduction in mold counts was less than 30%, but levels of detectable AFB1 were significantly reduced.

In the second study, we found a significant reduction in BW (when 2.5 µg/g AF diet was fed to broilers for 4 and 5 weeks) as compared to the control, which is consistent with the study by Miazzo et al. (2000), who reported a 11% body weight gain reduction at 2.5 µg/g of AF supplemented in the feed. However, we observed a reduction of BW due to AF exposure in birds only after 28 days of the experimental period (Fig 3). In addition, we did not observe any effect of AF on the feed intake and FCR in chickens, which concurred with the findings of Maizzo et al. (2000) and Pimpukdee et al. (2004). The effects of AF on feed intake and FCR are not always consistent because they depend on the composition of experimental diets, particularly different protein sources and levels, which were reported to alter protein utilization and animal response to AF in poultry (Richardson et al., 1987; Coffey et al., 1989).

The toxicity of AF is initiated through bioactivation to its toxic intermediates, which is mediated by cytochrome P450 enzymes located in the liver, thus making the liver the primary target for AF toxicity (Wogan, 1999; Wild and Turner, 2002). Furthermore, it has been documented that the negative effects of AF on chicken performance might be attributed to its harmful effects on liver weight and the liver function. In the current study, we observed a significantly increased relative liver weight in chickens fed with 2.5 µg/g AF as compared to the control group (Table 1). The enlarged liver may be due to the fatty infiltration and tissue proliferation (Eraslan et al., 2004; Shi et al., 2009) brought about by AF. In addition, it has been suggested that AF may generate a more profound toxicosis in modern broilers because of the rapid growth that requires faster hepatic metabolism (Yunus et al., 2011). In line with these observations, histopathological analysis of the enlarged liver collected from the AF-treatment group revealed severe necrosis, bile duct proliferation, and hypertrophied liver cells as compared to the control. However, these lesions were found to be alleviated in the liver samples from phytochemical-treated groups (Fig. 4). Thus, the findings from this study suggest the potential ability of CR and TC to counteract the harmful effects of AF, especially in reducing hepatic hispathological changes induced by the toxin.

Apart from the toxicity of AF on the liver, the immunosuppressive nature of AF is another well-documented adverse effect on birds (Yunus et al., 2011). Aflatoxin consumption has been reported to cause vaccine failure (Mohiuddin and Reddy, 1993) due to AF-induced decrease in antibody titers against Newcastle disease vaccine (Mohiuddin and Reddy, 1993). Verma et al. (2004) found a decreased relative weight of bursa of Fabricius from the chicken fed with 2.0 µg/g AF. In our study, results showed that the lymphoid organ, bursa of Fabricius of chickens given AF were markedly reduced in size, whereas CR and TC supplementation in the presence of AF significantly improved the relative bursa of Fabricius weight in week 4 when compared to AF-alone group (Table 2).

The mechanism of action of CR and TC against AF-induced toxicity is not yet documented. However, Ramirez et al. (2012) reported that CR was able to inhibit the activity of P450 enzymes in vitro. Since the toxicity of AF is initiated from a complex metabolism by P450 enzymes in the liver, the reduced activity of P450 might be one of the potential protective mechanisms of phytochemicals to reduce aflatoxicosis in chickens. In addition, CR and TC are known to be potent antioxidants that can act as a scavenger of free radicals (Chen et al., 2009) and can influence the activities of enzymes that are associated with AF detoxification. For example, Gowder and Devaraj (2006) observed that the activity of glutathione S-transferase (GST) was significantly increased in rats that were orally given cinnamaldehyde for 90 days. The increase in the level of GST might potentially increase the detoxification of AF-related toxic intermediates during AF metabolism in the liver and increase the excretion of toxic AF intermediates through urine and bile (Wild and Turner, 2002). However, these findings need to be validated in chickens.

In general, the commercial poultry feed is subjected to high temperatures to form pellets, and pelleting improves both feed handling and bird performances (Leeson and Summers, 1991). Therefore, the heat stability of phytochemicals is important to ensure their efficacy in improving feed safety. Two temperatures (150oF and 180oF) were used in the study, which were the conditioning temperatures used for the heat sensitive ingredients and the regular ingredients, respectively, according to the commercial manufacturing processes of poultry feed (Anonymous, 2003; Abdollahi et al., 2013). Results of the study showed no significant difference between the recovery rate (%) of CR and TC in the feed either treated at 150oF/ 180oF or untreated feed samples throughout the entire storage period (Fig. 5).

Table 1. Effect of in-feed supplementation of CR and TC on relative liver weight of chickens fed with 2.5 µg/g AF. CR: carvacrol; TC: trans-cinnamaldehyde; AF: aflatoxins

Table 2. Effect of in-feed supplementation of CR and TC on relative bursa of Fabricius weight of chickens fed with 2.5 µg/g AF. CR: carvacrol; TC: trans-cinnamaldehyde; AF: aflatoxins

Figure 1. Effect of carvacrol (CR) at 0.4, 0.8, and 1.0% on Aspergillus flavus NRRL 3357 and Aspergillus parasiticus NRRL 4123 growth and aflatoxin production in poultry feed

Figure 2 Effect of trans-cinnamaldehyde (TC) at 0.4, 0.8, and 1.0% on Aspergillus flavus NRRL 3357 and Aspergillus parasiticus NRRL 4123 growth and aflatoxin production in poultry feed

Figure 3. Effect of in-feed supplementation of carvacrol (CR) and trans-cinnamaldehyde (TC) on body weight of chickens fed with 2.5 µg/g AF

Figure 4. Effect of in-feed supplementation of carvacrol (CR) and trans-cinnamaldehyde (TC) on liver histopathology in chickens fed with 2.5 µg/g AF in week 3

Figure 5. Heat stability of carvacrol (CR) and trans-cinnamaldehyde (TC) in chicken feed

References:

Abdollahi, M. R., V. Ravindran, and B. Svihus, B. (2013). Pelleting of broiler diets: An overview with emphasis on pellet quality and nutritional value. Animal feed science and technology, 179(1), 1-23.

Anonymous. 2003. Journal of Feed Mix (7):9-10. http://www.allaboutfeed.net/PageFiles/10186/001_boerderij-download-AAF10419D01.pdf

Bluma R. V. and M. G. Etcheverry. (2006) Influence of Bacillus spp. isolated from maize agroecosystem on growth and aflatoxin B1 production by Aspergillus section Flavi. Pest Manag. Sci. 62:242-251.

Chen, F., Shi, Z., Neoh, K. G., and Kang, E. T. (2009) Antioxidant and antibacterial activities of eugenol and carvacrol‐grafted chitosan nanoparticles. Biotechnol Bioeng 104.1:30-39.

Coffey, M. T., Hagler, W. M., and Cullen, J. M. (1989) Influence of Dietary Protein, Fat or Amino Acids on the Response of Weanling Swine to Aflatoxin B J Anim Sci 67.2:465-472.

Eraslan, G. O. K. H. A. N., Akdogan, M. E. H. M. E. T., Yarsan, E. N. D. E. R., Essiz, D. I. N. C., Sahindokuyucu, F. A. T. M. A., Hismiogullari, S. E., and Altintas, L. E. V. E. N. T. (2004) Effects of aflatoxin and sodium bentonite administered in feed alone or combined on lipid peroxidation in the liver and kidneys of broilers. Bull Vet Inst Pulawy 48.3:301-304.

Gowder, S. J., and Devaraj, H. (2006) Effect of the food flavour cinnamaldehyde on the antioxidant status of rat kidney. Basic Clin Pharmacol Toxicol 99.5:379-382.

Kusumaningtyas, E., R. Widiastuti, and R. Maryam. (2006) Reduction of aflatoxin B1 in chicken feed by using Saccharomyces cerevisiae, Rhizopus oligosporus, and their combination. Mycopathologia. 162:307-311.

Leeson, S., J. D. Summers, and L. J. Caston, L. J. (1991). Diet dilution and compensatory growth in broilers. Poul. Sci. 70(4), 867-873.

Miazzo, R., Rosa, C. A. R., Carvalho, E. D. Q., Magnoli, C., Chiacchiera, S. M., Palacio, G., Saenz M. Basaldella E., and Dalcero, A. (2000) Efficacy of synthetic zeolite to reduce the toxicity of aflatoxin in broiler chicks. Poul Sci 79.1:1-6.

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Research conclusions:

According to the results from the heat stability of the phytochemicals treated with commercial manufacturing temperatures in the chicken feed, we observed no significant difference between the untreated control and the heat-treated groups (both 150oF and 180oF), which supports the use of the phytochemicals as feed additives in chicken feed to control AF contamination and subsequent aflatoxicosis in chickens. The findings in this study demonstrated that phytochemicals at 0.4%, 0.8%, and 1% significantly inhibited the growth of the aflatoxin-producing molds as well as the production of aflatoxins in the chicken feed for up to three months. In addition, in-feed supplementation of CR and TC at 0.75% reduced aflatoxicosis in chicken by improving bird performance and reducing AF-induced toxicity in liver. The results suggest that CR and TC could potentially be used as feed additives to control chicken aflatoxicosis.

Participation Summary

Education & Outreach Activities and Participation Summary

Participation Summary

Education/outreach description:

The results of this study were presented at the Poultry Science Association annual meetings in July 2015 at Louisville, KY and in July 2016 at New Orleans, LA, SARE-GNE15-113. The manuscripts are also being prepared for submission to Poultry Science and Journal of Mycotoxin Research. In addition, we will communicate the results of our study to feed companies and poultry producers through extension conferences and meetings such as the Connecticut Poultry Association. We will also include our results on UConn’s Poultry Pages website and the Connecticut Poultry Association website.

Project Outcomes

Project outcomes:

The economic losses due to AF contamination to the US poultry industry exceed $143 million annually. The findings of the current work suggest that CR and TC could potentially be used as feed additives to control the contamination of AF-producing molds and AF, which will benefit the profitability of the poultry industry. Especially due to the lipophilic nature of the phytochemicals, CR and TC can be easily mixed with other feed ingredients in a poultry ration. The cost of TC is ~ $2/lb, whereas CR is reported to cost ~ $22/lb. However, the cost of these chemicals in bulk quantities is expected to be significantly lower. Therefore, CR and TC could practically be used as ingredients in poultry feed to control aflatoxicosis, especially in light of the observed anti-toxigenic effect at concentrations as low as 0.4% in the feed.

Farmer Adoption

Currently, the farmers use AF-binding adsorbents such as hydrated sodium calcium aluminosilicates (HSCAS) in chicken feed to control AF contamination. However, there may be certain risk factors of the inclusion in diet before properly tested since several adsorbents have been shown to impair nutrient utilization and mineral absorption in animals. Previously, our lab has confirmed that in-feed supplementation of CR and TC at 0.75% did not significantly affect the essential mineral content, including calcium, phosphorus, magnesium, potassium, sodium, manganese, zinc, copper, iron. These findings were presented at the Poultry Science Association annual meetings in July 2015 at Louisville, KY and some farmers had expressed their interests to the results of the study.

Assessment of Project Approach and Areas of Further Study:

Areas needing additional study

While the results of this study revealed that CR and TC could potentially be used as feed additives to control chicken aflatoxicosis; however, follow up studies under field conditions using a large of number of birds are warranted.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.