Symrise – Carvacrol by Ross Whitehead

Carvacrol For use in animal nutrition

Carvacrol For use in animal nutrition Introduction

Symrise Carvacrol

Ever since their discovery as a growth and performance improving agent, antibiotics have been used regularly in animal feed to increase the conversion efficiency and lower morbidity/ mortality rates.

Carvacrol is one of the active chemical components of oregano, thyme, and marjoram essential oils. These essential oils contain between 5% and 80% carvacrol. Carvacrol has the characteristic pungent and warm odor of oregano. Literature data indicates that carvacrol contains antimicrobial and antioxidant functions both when used alone and in combination with other products like cinamaldehyde or thymol.

Overuse of antibiotics in modern animal production facilities promotes the emergence of antibiotic-resistant pathogens. Increasing antimicrobial resistance limits treatment options and increases the number, severity, and duration of microbial infections. Increasing cases of antibiotic resistant bacteria have brought concerns from the public and from health officials. The European Union was the first region to take action in an effort to try to decrease the proliferation of antibiotic resistant bacteria and in 2006 the EU banned the use of antibiotics in animal feed for non-essential purposes. In 2013, the US Food and Drug Administration put into place a policy to phase out the use of antibiotics as growth agents in animals raised for meat within the next 3 years. The industry is poised to explore alternatives to antibiotics to maintain or to increase the efficiency of conversion and to control mortality rates. Phytochemicals such as Carvacrol have been shown (in vitro) to be a promising antimicrobial alternative inhibiting the growth of bacterial strains such as; E.coli, B.cereus, and P. aeruginosa. Used in combination with other phytochemicals such as thymol and cinamaldehyde, carvacrol may be an effective alternative for antibiotics in animal feed.

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Symriseâ&#x20AC;&#x2122;s Carvacrol (Phenol, 2-methyl-5-(1methylethyl)-, CAS# 499-75-2, FEMA# 2245) is derived from l-Carvone which, in turn, is manufactured from a sustainable citrus d-limonene using a proprietary green process. It is a high purity product which comprises at least 99.5% carvacrol. This is a multifaceted compound that can be used as a flavor and one which may present antibacterial and antioxidant properties.

Carvacrol Independent Clinical Studies on Carvacrol Effects of carboxy methyl cellulose and thymol + carvacrol on performance, digesta viscosity and some blood metabolites of broilers

Rearing conditions influence nutrient availability of plant extracts supplemented diets when fed to broiler chickens.

H. Hashemipour*, H. Kermanshahi, A. Golian, V. Khaksar Journal of Animal Physiology and Animal Nutrition. 2013 Sep 16. doi: 10.1111/jpn.12121. ...............................................

Pirgozliev V, Bravo D, Rose SP. Journal Animal Physiology Animal Nutrition. 2013 Sep 5. doi: 10.1111/jpn.12119. ...............................................

This study was conducted to evaluate the effects of thymol + carvacrol as plant essential oils on performance, digesta viscosity and some blood metabolites of broilers fed diets supplemented with carboxy methyl cellulose (CMC) and/or thymol + carvacrol. In a completely randomized design with a 2 Ă&#x2014; 3 factorial arrangement, two levels of CMC (0% and 2%) and three levels of thymol+carvacrol (0, 100 and 200 mg/ kg) were used. Each of the six dietary treatments was fed to five replicate pens of 12 birds each from 0 to 42 days of age. Body weight gain (BWG), feed intake, feed conversion ratio (FCR), intestinal digesta viscosity and pH, plasma lipids and some blood metabolites were recorded. The inclusion of 2% CMC decreased (p < 0.05) BWG by 2.2% and increased FCR by 2.3% at 42 days of age. Carboxy methyl cellulose significantly increased the digesta viscosity and decreased serum total cholesterol, but had no significant effect on triglyceride, pH, HDL, LDL and other blood metabolites measured in this experiment. Thymol + carvacrol had no effect on feed intake, but significantly increased (p < 0.05) BWG from 1 to 42 days of age and improved FCR (p < 0.05) by the addition of 100 and 200 mg/kg thymol + carvacrol respectively. Inclusion of thymol + carvacrol at levels of 100 and 200 mg/ kg in the diets decreased digesta viscosity and serum total cholesterol (p < 0.05) and also increased AST at a 200 mg/ kg thymol+carvacrol without any effect on creatine kinase (CK). Thymol + carvacrol significantly increased total protein (TP), albumin and globulin (p < 0.05). In conclusion, CMC at an inclusion level of 2% of diet increased digesta viscosity and reduced growth performance. However, thymol + carvacrol decreased digesta viscosity and consequently improved the performance of broilers fed the CMC-based diet. Therefore, thymol + carvacrol addition to viscose-based diets might be helpful to alleviate the negative effects of viscous compounds in poultry diets.

The effects of a standardised mixture of essential oils, including 5% carvacrol, 3% cinnamaldehyde and 2% capsicum (XT 6930; Pancosma S.A), on dietary apparent metabolisable energy corrected for nitrogen retention (AMEn), nutrient digestibility and mucin secretions, measured as sialic acid (SA) were investigated in broilers fed on the same diet but reared under different conditions, that is, cages and floor pens littered with wood shavings used in previous broiler study. The use of XT reduced (p < 0.05) nitrogen digestibility (0.585 vs. 0.544) and tended (p = 0.072) to reduce dry matter digestibility (0.733 vs. 0.717) of the diet when fed to birds reared in cages. However, XT supplementation improved (p < 0.05) fat digestibility (0.844 vs. 0.862) and tended (p = 0.093) to increase AMEn (14.01 vs. 14.25 MJ/ kg DM) of the same diet when fed to broilers reared in floor pens. Essential oils supplementation tended (p = 0.059) to increase the secretion of SA, when fed to birds reared in cages (11.24 vs. 14.18 Îźg), but did not influence (p > 0.05) the SA secretion from birds reared in floor pens. The results obtained from the cage study tend to be the opposite of those obtained from the floor pen study. This suggests that the efficiency of dietary plant extracts may be influenced by the rearing/hygienic conditions of poultry. Based on the overall results, it can be concluded that information on rearing conditions should be taken into account for more complete interpretation of the experimental data emanating from experiments involving use of essential oils typified by those considered in this study.

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Carvacrol Independent Clinical Studies on Carvacrol Effect of thymol and carvacrol feed supplementation on performance, antioxidant enzyme activities, fatty acid composition, digestive enzyme activities, and immune response in broiler chickens.

Effect of plant derived antimicrobials on Salmonella Enteritidis adhesion to and invasion of primary chicken oviduct epithelial cells in vitro and virulence gene expression.

Hashemipour, H.; Kermanshahi, H.; Golian, A.; Veldkamp, T. Poultry Science. Aug 2013, Vol. 92 Issue 8, p2059-2069. 11p. DOI: 10.3382/ps.2012-02685. ...............................................

Upadhyaya I, Upadhyay A, Kollanoor-Johny A, Darre MJ, Venkitanarayanan K. Int J Mol Sci. 2013 May 21;14(5):10608-25. ...............................................

This trial was conducted to evaluate the effects of dietary supplementation of phytogenic product containing an equal mixture of thymol and carvacrol at 4 levels (0, 60, 100, and 200 mg/kg of diet) on performance, antioxidant enzyme activities, fatty acid composition, digestive enzyme activities, and immune response in broiler chickens.

Salmonella Enteritidis (SE) is a major foodborne pathogen in the United States and one of the most frequently reported Salmonella serotypes globally. Eggs are the most common food product associated with SE infections in humans. The pathogen colonizes the intestinal tract in layers, and migrates to reproductive organs systemically. Since adhesion to and invasion of chicken oviduct epithelial cells (COEC) is critical for SE colonization in reproductive tract, reducing these virulence factors could potentially decrease egg yolk contamination. This study investigated the efficacy of subinhibitory concentrations of three plant-derived antimicrobials (PDAs), namely carvacrol, thymol and eugenol in reducing SE adhesion to and invasion of COEC, and survival in chicken macrophages. In addition, the effect of PDAs on SE genes critical for oviduct colonization and macrophage survival was determined using real-time quantitative PCR (RT-qPCR). All PDAs significantly reduced SE adhesion to and invasion of COEC (p < 0.001). The PDAs, except thymol consistently decreased SE survival in macrophages (p < 0.001). RT-qPCR results revealed down-regulation in the expression of genes involved in SE colonization and macrophage survival (p < 0.001). The results indicate that PDAs could potentially be used to control SE colonization in chicken reproductive tract; however, in vivo studies validating these results are warranted.

Each of the 4 diets was fed to 5 replicates of 12 chicks each from d 0 to 42. The inclusion of thymol + carvacrol linearly decreased (P < 0.05) feed intake, but the highest {P < 0.05) BW gain (ADG) and feed efficiency was observed in broilers offered 200 mg/kg of phytogenic product. The phytogenic product linearly increased (P < 0.05) superoxide dismutase and glutathione peroxidase activities and decreased (P < 0.05) malondialdehyde level in thigh muscle at d 42 and serum and liver at d 24 and 42. Total saturated fatty acids were depressed (P < 0.05) and total polyunsaturated fatty acid and n-6 were linearly increased (P < 0.05) in serum and thigh by the inclusion of phytogenic product compared with the control diet. Supplementation with thymol + carvacrol also increased intestinal and pancreatic trypsin, lipase, and protease activities in 24-d-old (linear, P < 0.05) but not in 42-d-old birds. thymol + carvacrol modified (linear, P < 0.05) immune response by increasing hypersensitivity response, total and IgG antisheep red blood cell titers, and decreasing heterophil to lymphocyte ratio compared with the control group. However, hemato-logical parameters and lymphoid organ weight were not affected by thymol + carvacrol. Thus, feed supplementation with thymol + carvacrol enhanced performance, increased antioxidant enzyme activities, retarded lipid oxidation, enhanced digestive enzyme activities, and improved immune response of broilers.

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Independent Clinical Studies on Carvacrol In vitro sensitivity of poultry Brachyspira intermedia isolates to essential oil components and in vivo reduction of Brachyspira intermedia in rearing pullets with cinnamaldehyde feed supplementation. Verlinden M, Pasmans F, Mahu M, Vande Maele L, De Pauw N, Yang Z, Haesebrouck F, Martel A. Poult Sci. 2013 May;92(5):1202-7. doi: 10.3382/ ps.2012-02690. ............................................... Cecal enteritis due to Brachyspira infections tends to be chronic in laying hens. Limited availability of antimicrobial drugs for use in laying hens emphasizes the need for alternative control measures. A broth microdilution method was used to determine the antimicrobial susceptibility of 20 Brachyspira intermedia field isolates from laying hen flocks to components of essential oils (EO). Minimal inhibitory concentration (MIC) distributions, obtained for 8 EO components, were all monomodal. Cinnamaldehyde had the lowest MIC values (40 to 80 mg/L), followed by nerolidol, capsaicin, carvacrol, and thymol (80 to 320 mg/L), eugenol (160 to 640 mg/L), and linalool (320 to 1,280 mg/L). The MIC ranges of piperine were mostly above the test range of 1,280 mg/L. In an in vivo experiment, coated transcinnamaldehyde was supplemented to the feed of rearing pullets. A completely randomized experimental design with 4 treatments and 3 replicates each (replicate = group of seven 1-d-old laying hen chickens) was applied. The negative and positive controls received a conventional feed during the whole trial. The positive controls were orally inoculated on 3 consecutive days (d 22, 23, and 24) with 1 mL of 1.0 × 10(8) cfu/mL of a B. intermedia field isolate. Two treatment groups (preventive and curative), identically inoculated, received the coated trans-cinnamaldehyde-supplemented feed (500 mg/kg of trans-cinnamaldehyde), the preventive group from d 1, the curative from d 25. On d 32, ceca were collected for bacteriologic Brachyspira enumeration. The mean enumeration of Brachyspira cells was decreased (P < 0.05) in the curative treated group versus the positive control group. The in vitro results of the present study demonstrate the potential of EO components as antimicrobials against poultry Brachyspira isolates, including isolates with acquired resistance for classic antimicrobial drugs. Reduction of

Brachyspira colonization in young pullets was obtained, in a curative way, in an in vivo study using feed supplemented with coated trans-cinnamaldehyde. Further studies are necessary to investigate the mode of action of the coated trans-cinnamaldehyde in reducing Brachyspira colonization of the ceca. A standardized combination of carvacrol, cinnamaldehyde and capsicum oleoresin improves performance and energy partitioning of broilers. Pirgozliev, V.; Bravo, D.; Rose, P.; Oguey, C.; Viel, B. Actes des 10èmes Journées de la Recherche Avicole et Palmipèdes à Foie Gras du 26 au 28 mars, 2013, La Rochelle, France 2013 pp. 711-715 ............................................... Previous research demonstrated that 100 g/t of a standardized blend of carvacrol, cinnamaldehyde and capsicum oleoresin (XT, XTRACT® 6930, Pancosma) was equivalent to 50 kcal/kg of metabolizable energy (ME). It was suggested that net energy for production (NEp) was a more appropriate parameter to assess the effect of phytonutrients on poultry, but no data is currently available. Consequently, the objective of this trial was to evaluate the effect of XT on performance, ME, NEp of broilers. Fours hundreds day old male broilers (Ross 308) were randomly allocated to 40 floor pens bedded with used litter. Treatments followed a 2*2 randomized complete block desing (10 animals/pen, 10 replications/treatment). First factor was the type of diet (based on corn-soybean or wheat-soybean meal), and the second factor was the level of XT (0 vs. 100 g/t). Feeds were given ad libitum for 21 days and formulated to be iso-proteic (21.5%), lower in ME (2890 kcal/kg) and slightly higher in non starch polysaccharides than the recommendations. Performance parameters (BWG, FI, FCR), apparent ME (AME), NEp and heat production (HP) were measured. The wheat based diet reduced (P<0.05) AMEand FCR, and increased (P<0.05) NEp. Whatever the cereal source, XT increased AME and NEp (P<0.05) and reduced FCR and HP (P<0.05). This suggests that XT induces a similar response when added to a diet based on corn or wheat. The relationship between energy parameters and performance was then assessed by linear regression. NEp was more highly correlated to Please see disclaimer at the end of this document.

Carvacrol Independent Clinical Studies on Carvacrol performance than AME. Therefore XT improves performance, AME and EP of diets fed to broilers. NEp seems to be a parameter more appropriate than AME to evaluate the effect of phytonutrients on broilers. Effect of essential oil compound on shedding and colonization of Salmonella enterica serovar Heidelberg in broilers. Alali WQ, Hofacre CL, Mathis GF, Faltys G. Poult Sci. 2013 Mar;92(3):836-41. doi: 10.3382/ ps.2012-02783. ............................................... The objectives of this study were to determine the effect of an essential oil blend (EO; carvacrol, thymol, eucalyptol, lemon) administered in drinking water on the performance, mortality, water consumption, pH of crop and ceca, and Salmonella enterica serovar Heidelberg fecal shedding and colonization in broiler birds following Salmonella Heidelberg challenge and feed withdrawal. Chicks were randomly assigned to water treatments containing 0.05, 0.025, or 0.0125% EO or untreated controls. Treatments were administered in drinking water on 0 to 7 and 35 to 42 d. One-half of the chicks were challenged with Salmonella Heidelberg and placed in pens with unchallenged chicks on d 1. Performance, mortality, water consumption, and pH were determined during the 42-d study. Prevalence of Salmonella Heidelberg was determined on drag swabs (0, 14, and 42 d) and in the ceca and crops (42 d). The 0.05% EO administered in drinking water significantly (P < 0.05) reduced Salmonella Heidelberg colonization in crops of challenged birds, significantly lowered the feed conversion ratio, and increased weight gain compared with controls. The 0.025% and 0.015% EO in drinking water significantly lowered the feed conversion ratio and increased weight gain compared with controls, but did not significantly reduce Salmonella Heidelberg colonization in the crops. The EO in drinking water did not significantly reduce Salmonella Heidelberg colonization in ceca or fecal shedding in broilers. The EO used in the study may control Salmonella Heidelberg contamination in crops of broilers when administered in drinking water and therefore may reduce the potential for cross-contamination of the carcass when the birds are Please see disclaimer at the end of this document. 6 processed.

Sub-lethal levels of carvacrol reduce Salmonella Typhimurium motility and invasion of porcine epithelial cells. Inamuco J, Veenendaal AK, Burt SA, Post JA, Tjeerdsma-van Bokhoven JL, Haagsman HP, Veldhuizen EJ. Vet Microbiol. 2012 May 25;157(1-2):200-7. doi: 10.1016/j. vetmic.2011.12.021. Epub 2011 Dec 24. ............................................... The European ban on the use of antibiotic growth promotors has increased the search for new alternatives to prevent pig intestinal microbial diseases and to stimulate growth. The addition of essential oils or components thereof, such as carvacrol, to pig feed is a promising alternative. In this report we determined the effect of sub-lethal concentrations of carvacrol on Salmonella Typhimurium. At concentrations where growth of Salmonella was not inhibited, carvacrol completely inhibited motility of the bacterium. This loss of motility was not due to the loss of the flagellum or to ATP shortage upon carvacrol treatment. Adhesion of Salmonella to IPEC-J2, porcine intestinal epithelial cells, was not affected by carvacrol but invasion was significantly reduced. In addition, the epithelial gene expression of porcine Î˛-defensin 2, an innate immune response to Salmonella infection, was reduced when Salmonella was exposed to carvacrol. This indicates that invasion but not adhesion of Salmonella triggers the porcine Î˛-defensin 2 expression of porcine epithelial cells. Influence of dietary thymol and carvacrol preparation and/or an organic acid blend on growth performance, digestive organs and intestinal microbiota of broiler chickens. Hasan Akyurek and Adem Yel. African Journal of Microbiology Research Vol. 5(8), pp. 979-984, 18 April, 2011 ............................................... This experiment was designed to investigate the effects of supplementation with plant extract, either alone or in combination with an organic acid on growth performance, intestinal organ measurements and intestinal microbiology. One-day-old male Ross 308 strain broiler chicken (n=96)

Independent Clinical Studies on Carvacrol were allocated to 4 dietary treatments in a randomized complete block design. Dietary treatments were: (i) basal diet (as a control) (C), (ii) basal diet + organic acid mixture feed (OA), (iii) basal diet + plant extract (PE) and (iv) basal diet + organic acid mixture + plant extract feed (OA+PE). Body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR) were not improved by supplementation of OA or PE to the diets. Proventriculus, gizzard, hearth, liver, pancreas, abdominal fat and bursa of Fabricious weights were not significantly affected by dietary treatments. OA and PE diets resulted in increased weight and length of duodenum, jejenum, ileum and caceum. However, differences between treatments were not statistically significant (P>0.05). Supplementation of organic acid has positive effect on ileal microbiology. In ileal digesta, LAB and yeast counts were significantly (P<0.001) increased for birds fed OA and PE, whereas Escherichia coli counts were significantly (P<0.001) decreased in OA group. In conclusion, as OA and PE supplementation had any positive effects on the performance. Ileum microflora of OA supplemented group changed for the benefit of non-pathogenic bacteria, probably due to the decrease in pH levels of ileum.Â E. coli count was found lower for OA treatment than the other groups. These results indicate that the OA can improve gut health. The products that researched show promising effects, however, further researchers may be useful to understand their effects better. The effect of feeding essential oils on broiler performance and gut microbiota. Tiihonen K, Kettunen H, Bento MH, Saarinen M, Lahtinen S, Ouwehand AC, Schulze H, Rautonen N. British Poultry Scence. 2010 Jun;51(3):381-92. ............................................... In this study the effect of a blend of essential oils (EO) comprising 15 g/tonne thymol and 5 g/tonne cinnamaldehyde on the performance and intestinal microbiota of broilers was investigated. 2. A total of 720 male Ross broilers were divided into two dietary treatments with 12 replicate pens per treatment. Broilers were given a control soybean-wheat-based diet with or without added EO in two diet phases (0-21 d and 22-42 d). 3. The blend

of EO increased body weight gain of broilers from 0 to 42 d by 45%. 4. Caecal microbiota were affected by the EO blend; in particular increases in the proportions of Lactobacillus and Escherichia coli at 41 d was observed. 5. The EO blend had major effects on caecal metabolites. The proportion of caecal butyrate at 20 and 41 d of age increased, whereas the proportion of caecal acetic acid at 20 d, and propionic acid and isovaleric acid at 41 d, decreased with the EO blend. In addition, the caecal proportion of spermine increased and tyramine decreased at 41 d of age with the EO treatment. 6. The present study shows that EO supplementation exerts a positive effect on intestinal microbiota with a concomitant enhancement in growth performance. The study suggests that modulation of broiler gut microbiota composition and activity through the administration of EO offers an effective means for improving broiler performance. Effects of dose and formulation of carvacrol and thymol on bacteria and some functional traits of the gut in piglets after weaning. Michiels J, Missotten J, Van Hoorick A, Ovyn A, Fremaut D, De Smet S, Dierick N. Arch Anim Nutr. 2010 Apr;64(2): 136-54. ............................................... Two trials were conducted to study the effects of dose and formulation of carvacrol and thymol on bacterial counts, metabolites and functional traits of the gut in weaned piglets. In the first experiment (Exp. I), 25 piglets (28 d, 6.59 +/- 0.48 kg BW) were allocated to five dietary treatments: a control diet, or the same diet supplemented with either carvacrol or thymol at doses of 500 and 2000 mg kg(-1). In the second experiment (Exp. II), 35 piglets (28 d, 7.99 +/0.73 kg BW) were assigned to seven dietary treatments: the same control diet as in Exp. I, or this diet supplemented with thymol in one of three formulations (on celite, on alphacel or microencapsulated) at doses of 500 and 2000 mg kg(-1). At 11/12 days post-weaning piglets were euthanised, and digesta from stomach, proximal and distal small intestine were sampled for bacteriological and biochemical analysis. Small intestinal tissue was sampled for histomorphological determinations. In none of the experiments or sections of the Please see disclaimer at the end of this document.

Carvacrol Independent Clinical Studies on Carvacrol gut was the number of bacteria lowered by the carvacrol or thymol supplementation. In Exp. I, the villus/crypt ratio at the distal small intestine for the experimental diets (1.30-1.32) was higher than for the control diet (1.24) (p < 0.05). Thymol fed animals in Exp. II had a lower number of intra-epithelial lymphocytes at the proximal (p < 0.05) and at the distal (p < 0.1) small intestine as compared to control animals. Mean concentration of the active ingredient in the stomach and proximal small intestine for the 2000 mg kg(-1) carvacrol diet was 521 and 5 mg kg(-1) fresh digesta, respectively, and for the 2000 mg kg(-1) thymol diets it ranged between 475 and 647 and between 13 and 24 mg kg(-1) fresh digesta, respectively. Cumulative absorption in the proximal small intestine was higher than 90% for all treatments and was not affected by formulation type. These data suggest that carvacrol and thymol can improve gut health, but evidence for clear antimicrobial effects towards the major culturable bacteria of the pig foregut is limited. Effects of thymol and carvacrol feed supplementation on lipid oxidation in broiler meat. Luna A, Lรกbaque MC, Zygadlo JA, Marin RH. Poultry Science 2010 Feb;89(2):366-70.doi: 10.3382/ ps.2009-00130. ............................................... Poultry meat is particularly prone to oxidative deterioration due to its high concentration of polyunsaturated fatty acids. The present study evaluates the effects of thymol and its isomer carvacrol on lipid oxidation when supplemented to the feed. Supplementation with the antioxidant butylated hydroxytoluene was used as a positive control. Thus, broiler chickens were assigned to 1 of 4 treatments: vehicle (control), 150 mg/kg of butylated hydroxytoluene (positive control), 150 mg/kg of thymol, or 150 mg/kg of carvacrol. Breast and thigh samples were taken at 0, 5, and 10 d of 4 degrees C storage. Lipid oxidation was determined by the analysis of 2-TBA reactive substances (TBARS). Sample storage for 5 to 10 d significantly increased the levels of TBARS. Feed supplementation did not significantly affect breast sample

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oxidation. However, after 5 and 10 d of storage, increasingly higher values of TBARS were detected in thigh samples of the control group in comparison to the 3 supplemented groups. Interestingly, the same lower values of TBARS were detected between those feed-supplemented groups. Therefore, the application of the natural antioxidants thymol or carvacrol could be useful to improve poultry meat quality. High-throughput gene expression analysis of intestinal intraepithelial lymphocytes after oral feeding of carvacrol, cinnamaldehyde, or Capsicum oleoresin. Kim DK, Lillehoj HS, Lee SH, Jang SI, Bravo D. Poultry Science 2010 Jan;89(1):68-81. ............................................... Among dietary phytonutrients, carvacrol, cinnamaldehyde, and Capsicum oleoresin are well known for their antiinflammatory and antibiotic effects in human and veterinary medicine. To further define the molecular and genetic mechanisms responsible for these properties, broiler chickens were fed a standard diet supplemented with either of the 3 phytochemicals and intestinal intraepithelial lymphocytes were examined for changes in gene expression by microarray analysis. When compared with chickens fed a nonsupplemented standard diet, carvacrol-fed chickens showed altered expression of 74 genes (26 upregulated, 48 downregulated) and cinnamaldehyde led to changes in the levels of mRNAs corresponding to 62 genes (31 upregulated, 31 downregulated). Most changes in gene expression were seen in the Capsicum-fed broilers with 98 upregulated and 156 downregulated genes compared with untreated controls. Results from the microarray analysis were confirmed by quantitative real-time PCR with a subset of selected genes. Among the genes that showed >2.0-fold altered mRNA levels, most were associated with metabolic pathways. In particular, with the genes altered by Capsicum oleoresin, the highest scored molecular network included genes associated with lipid metabolism, small molecule biochemistry, and cancer. In conclusion, this study provides a foundation to further investigate specific chicken genes that are expressed in response to a diet containing carvacrol, cinnamaldehyde, or Capsicum oleoresin.

Independent Clinical Studies on Carvacrol Influence of diet type on the inclusion of plant origin active substances on morphological and histochemical characteristics of the stomach and jejunum walls in chicken. Jamroz D, Wertelecki T, Houszka M, Kamel C. J Anim Physiol Anim Nutr (Berl). 2006 Jun;90(5-6):255-68. ............................................... Three hundred and thirty-six 1-day-old male Hubbard HI-Ye broiler hybrids, kept in battery cages, were fed with diets based on maize (groups I and II) or wheat and barley (groups III and IV) and supplemented with or without plant extract (XT* 100 mg/kg) containing 5% carvacrol, 3% cinnamaldehyde and 2% of capsicum oleoresin. The morphological and histochemical examinations were carried out on days 21 and 42 of bird’s age. The middle part of glandular part of the stomach and 30 mm long segment from the central part of the small intestine (jejunum) were taken out and then prepared for morphometrical and histochemical assays. Mobilization of mucocytes in superficial epithelium of the glandular stomach and increased secretion of neutral mucopolysaccharides and small amounts of sialomucins with or without local cell disruption with releasing of large amounts of mucus were observed in both ‘grain’ groups of 21-day-old birds fed with extract. In some animals, particularly those fed mixtures with plant extract, the folds of the proventriculum mucosa were fused into large, unshaped structures. In groups fed with plant extract the mucus secretion intensity and accumulation inside cells of the gastrointestinal mucosa were slightly higher. Morphological changes on gastrointestinal mucosa observed in young chickens fed XT were reduced in older animals. The results of this study showed that the increased releasing of large amounts of mucus and the creation of a thick layer of mucus on glandular stomach and wall of jejunum in chickens fed diets with plant extract could suggest villi-related protective properties of the use of the carvacrol, cinnamaldehyde and capsaicin mixture. This can explain the reduced possibility of adhesion to epithelium and number of Escherichia coli, Clostridium perfringes and fungi in the intestinal content of bird fed with XT supplemented diet. In morphometrical parameters of depth of jejunum crypt and height of villi, the influence of kind of grain and extract supplementation was observed in 21-day-old chickens only. The significant interaction between higher jejunum wall villi layer was

observed only in chickens fed on maize diet supplemented with plant extract. Effects of feed additives and mixed eimeria species infections on intestinal microbial ecology of broilers M. E. Hume,*2 S. Clemente-Herna´ndez,†‡ and E. O. OviedoRondo´n†3 Poultry Science 2006 85:2106-2111 ............................................... Evaluation of digestive microbial ecology is necessary to understand effects of growth-promoting feed. In the current study, the dynamics of intestinal microbial communities (MC) were examined in broilers fed diets supplemented with a combination of antibiotic (bacitracin methylene disalicylate) and ionophore (Coban 60), and diets containing 1 of 2 essential oil (EO) blends, Crina Poultry (CP) and Crina Alternate (CA). Five treatments were analyzed: 1) unmedicated uninfected control; 2) unmedicated infected control; 3) feed additives monensin (bacitracin methylene disalicylate) + monensin (Coban 60; AI); 4) EO blend CP; and 5) EO blend CA. Additives were mixed into a basal feed mixture, and EO were adjusted to 100 ppm. Chicks were infected by oral gavage at 19 d of age with Eimeria acervulina, Eimeria maxima, and Eimeria tenella. Duodenal, ileal, and cecal samples were taken from 12 birds per treatment just before and 7 d after challenge; 2 samples each were pooled to give a final number of 6 samples total; and all pooled samples were frozen until used for DNA extraction. Denaturing gradient gel electrophoresis was used to examine PCRamplified fragments of the bacterial 16S ribosomal DNA variable region. Results are presented as percentages of similarity coefficients (SC). Dendrograms of PCR amplicon or band patterns indicated MC differences due to intestinal location, feed additives, and cocci challenge. Essential oil blends CP and CA affected MC in all gut sections. Each EO had different effects over MC, and they differed in most instances from the AI group. The cocci challenge caused drastic MC population shifts in duodenal, ileal, and cecal sections (36.7, 55.4, and 36.2% SC, respectively). Diets supplemented with CP supported higher SC between pre- and postchallenge MC (89.9, 83.3, and 76.4%) than AI (81.8., 57.4, and 60.0%). We concluded that mixed coccidia challenge caused drastic shifts in MC. Please see disclaimer at the end of this document.

Carvacrol Independent Clinical Studies on Carvacrol Effect of dietary dried oregano leaves on growth performance, carcase characteristics and serum cholesterol of female early maturing turkeys. Bampidis VA, Christodoulou V, Florou-Paneri P, Christaki E, Chatzopoulou PS, Tsiligianni T, Spais AB. Br Poult Sci. 2005 Oct;46(5):595-601. ............................................... A study was conducted with 120 female early maturing turkeys to test the effect of dietary dried oregano leaves (Origanum vulgare subsp. hirtum) on body weight (BW), feed intake (FI), feed conversion efficiency (FCE), carcase characteristics and serum cholesterol concentration. Dried oregano leaves had a content of 3.6 ml essential oils/100 g, while the carvacrol content was 855 g/kg of the total essential oils. 2. From 1 to 84 d of age, the turkeys were fed on 4 diets varying in oregano content (OR0, no oregano-control; OR45, 1.25 g oregano/kg; OR90, 2.5 g oregano/kg; OR135, 3.75 g oregano/kg). Birds were given feed and water ad libitum. 3. BW was unaffected by oregano throughout the experiment. FI and FCE were similar among all treatments until 42 d of age. From 43 to 84 d of age and for the overall experimental period, FI decreased linearly in treatment OR135 and FCE increased linearly with dietary oregano content. Body and carcase weights, carcase yield, and the relative weights of the heart and liver were not significantly affected by oregano content. The relative weights of the gizzard and small intestine decreased linearly with oregano content. Serum cholesterol content was similar among all treatments. 4. In the present study, dietary oregano (1.25, 2.5 and 3.75 g/kg) improved FCE in female early maturing turkeys between 43 and 84 d, with the lowest oregano inclusion (1.25 g/kg) giving the most cost effective diet. Thus, dried oregano leaves may be used as a natural herbal growth promoter for early maturing turkeys.

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Effect of plant extracts and formic acid on the intestinal equilibrium of early-weaned pigs. Manzanilla EG, Perez JF, Martin M, Kamel C, Baucells F, Gasa J. Journal Animal Science. 2004 Nov; 82(11):3210-8 ............................................... We evaluated the effects of a plant extracts mixture (XT) standardized in 5% (wt/wt) carvacrol, 3% cinnamaldehyde, and 2% capsicum oleoresin (oregano, cinnamon and Mexican pepper), alone or in combination with formic acid (FA), on the productive performance and the intestinal ecosystem of the early-weaned pig. Pigs weaned at 20 +/- 1 d of age (n = 216) were allocated in 24 pens and fed a standard medicated prestarter diet for 12 d. Twelve days after weaning, a stress management system based on social and dietary stress factors was applied to the animals, after which, each group was allocated to one of six dietary treatments, which followed a factorial arrangement, with three levels (as-fed basis) of the XT (0, 150, and 300 mg/kg) and two levels of FA (0 and 0.5%). On d 24 and 25 after the stress episode, eight pigs per treatment were killed to examine variables describing some aspects of the gastrointestinal ecology. Two days after the stress episode, an Escherichia coli K88 diarrhea episode occurred, and five casualties were registered. Four of the five deaths occurred in pens of pigs not fed the XT. The FA resulted in better G:F (P = 0.040) in coincidence with shorter villous height (P = 0.073) and lower rectal total microbial mass (P = 0.078). Both XT and FA addition increased stomach content (P = 0.006 and 0.003, respectively) and percentage of DM (P = 0.089 and 0.010, respectively), suggesting an increased gastric retention time; consequently, pH was also increased (P = 0.005 and 0.060, respectively). The XT decreased ileum total microbial mass (P = 0.025) and increased the lactobacilli:enterobacteria ratio (P = 0.002). The VFA profile in the cecum and colon was modified by XT inclusion, increasing the proportion of acetate (P = 0.018 and 0.025, respectively) and diminishing the proportion of butyrate (P = 0.096 and 0.040, respectively) and valerate (P = 0.001 and 0.039, respectively). Both XT and FA were shown to be effective in modifying the gastrointestinal ecosystem, stomach contents, and stomach emptying rate, which are proposed as important aspects in the mechanisms of action for these additives.

Independent Clinical Studies on Carvacrol Immunostimulatory effect of dietary oregano etheric oils on lymphocytes from growth-retarded, low-weight growing-finishing pigs and productivity.

The effect of two different blends of essential oil components on the proliferation of Clostridium perfringens in the intestines of broiler chickens.

Walter BM, Bilkei G. Tijdschr Diergeneeskd. 2004 Mar 15;129(6):178-81. ...............................................

Mitsch P, Zitterl-Eglseer K, Kรถhler B, Gabler C, Losa R, Zimpernik I. 2004 Poultry Sciences 83:669-675 ...............................................

The present study was designed to evaluate the possible effect of dietary oregano etheric oils as non-specific immunostimulating agents in growth-retarded, low-weight growing-finishing pigs. Forty-nine growth-retarded (> 10% under average weight in a group) growing-finishing pigs of the same age were assigned to two groups and treated as follows: Group 1 (n = 25): the animals weighed 58.2 +/- 2.4 kg and were fed until slaughter ad libitum with a commercial fattening diet supplemented with 3000 ppm commercial oregano feed additive (Oregpig Pecs, Hungary). Oregpig is dried leaf and flower of Origanum vulgare, enriched with 500 g/kg cold-pressed essential oils of the leaf and flower of Origanum vulgare. Analysis of Oregpig: 60 g carvacrol and 55 g thymol/kilogram. Group 2 (n = 24): the animals weighed 57.9 +/- 2.6 kg and were fed until slaughter with the same diet without Oregpig supplementation. Oregpig-receiving pigs showed a significantly (P < 0.05) better average daily gain and feed conversion rate than the non-treated animals (Oregpig group 788.1 +/- 31.3 g, control animals 709.3 +/- 42.2 g; 2.96, vs. 3.08, respectively). Mortality was significantly (P < 0.001) higher in the non-treated animals (Oregpig group, 1 animal = 4%; control, 8 animals = 33.3%). The proportion of CD4, CD8, MHC class II antigen, and non-T/ non-B cells in peripheral blood lymphocytes was significantly higher in the Oregpig-receiving pigs than in the control animals. The proportion of CD4+ CD8+ double-positive T lymphocytes in peripheral blood and mesenteric lymph nodes was higher in the Oregpig-receiving pigs than in the control animals. Implication: Dietary oregano improves growth in growth-retarded growing-finishing pigs and has non-specific immunostimulatory effects on porcine immune cells.

The effect of 2 different blends of essential oils on Clostridium perfringens (Cp) in the intestine and feces of broiler chickens was tested in 6 field trials for each blend. One hundred parts per million of the blends were mixed in a commercial corn-based diet throughout the entire growing period for experimental flocks. Samples from the jejunum, cecum, cloaca, and feces were taken on d 14, 21, and 30 from experimental and control flocks and tested quantitatively for Cp via blood agar plate, litmus milk medium, and ELISA. Blend A reduced (P < or = 0.05) the average Cp concentration in the feces on all sampling days, in the jejunum and cecum on d 14 and 21, and in the cloaca on d 14. Blend B effected a significant reduction of Cp concentration in the jejunum on d 14 and 30 and in the cloaca on d 14. The percentages of specimens from the control group that tested positive for Cp were 83.3% for feces, 88.0% for jejunum and cloaca, and 82.6% for cecum. Specimens from the feces and 3 sections of the intestine were Cp positive in groups treated with blend A (60.8, 64.6, 47.9, and 70.8%) and with blend B (65.9, 63.6, 63.6, and 72.7%). Our results indicate that specific blends of essential oil components can control Cp colonization and proliferation in the gut of broilers and therefore may be of help to prevent problems with Cp and necrotic enteritis.

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Carvacrol Independent Clinical Studies on Carvacrol Essential oils in broiler nutrition. Lee, K. –W., Everts, H., and Beyenen, A.C. International Journal of Poultry Science 3(12): 738-752, 2004 ............................................... Based on literature data it can be concluded essential oils originating from plants have anti-microbial activity and have toxic effects in poultry only when administered at very high doses. Antioxidant activity and hypocholesterolemic effects have been reported in chickens. In various studies, but not all, a growth enhancing effect of essential oils has been found. The characteristic flavor of essential oils might play role in poultry performance, but this needs to be confirmed. Essential oils may stimulate the digestion process. It appears that individual compounds of an essential oil have a wide range of activities and may act in an additive, synergistic and antagonistic fashion. The effect of essential oils in poultry may not only be confined to the microflora, but may extend to animal metabolism. Knowing the activity and effects of individual compounds is useful to formulate mixtures of compounds so as to enhance efficacy. In conclusion, dietary essential oils may be used as alternatives to antibiotics, but whether their effects on growth performance are a consequence of anti-microbial activity needs to be studied further. Carvacrol and thymol reduce swine waste odor and pathogens: stability of oils. Varel VH. Curr Microbiol. 2002 Jan;44(1):38-43. ............................................... An incomplete anoxic fermentation of livestock waste results in offensive odor emissions. Antimicrobial additives may be useful in controlling odor emissions and pathogens. Natural antimicrobial compounds, carvacrol or thymol at 16.75 mM (2.5 g/l) completely inhibited the production of the offensive odor compounds, isobutyrate, valerate, isovalerate, and cresol, and significantly reduced other short-chain volatile fatty acids and gas emissions from swine waste. Fecal coliforms were reduced from 6.3 x 10(6) to 1.0 x 10(3) cells per ml 2 days after treatment with carvacrol (13.3 mM) and were not detectable within 14 days. Total culturable 12

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anaerobic bacteria were reduced from 12.4 x 10(10) to 7.2 x 10(8) cells per ml after 2 days and were suppressed below this level for 28 days. Lactate production was not prevalent in untreated swine waste indicating that the microbial populations differ from those in cattle waste. Carvacrol and thymol were stable in swine waste under anoxic conditions for 62 days with 90 to 95% of the additive being recovered in the waste solids. In conclusion, carvacrol and thymol are not metabolized in anoxic swine waste and they are potentially useful in controlling odor emissions and pathogens in swine waste.

Patents Patent EP 1768491 A1 and A4 – Methods and compositions for controlling parasitic infections of animals. Inventors: D. Cepta; A. J. Connolly. Applicant: Alltech Patent 6,106,838 – Pharmaceutical compositions containing herbal-based active ingredients; methods for preparing same and uses of same for medical and veterinary purposes. Inventor: Fotios Nitsas Patent 6,322,825 – Compositions containing thymol and carvacrol and methods of treating gastrointestinal infections with the compositions. Inventor: Dusan Ninkov. Assignee: Ropapharm Patent EP 1207865A1 – Composition for treatments of infections of humans and animals. Inventor: Dusan Ninkov. Applicant: Van Beek Global/ Ninkov LLC Patent EP 2170100 A1 – Animal feed product. Applicant: Danisco Patent EP 1178736 – A composition containing carvacrol and thymol for use as a bactericide. Inventor: Riccardo Losa. Applicant: Akzo Nobel

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