Effects of organic acids or natural plant extracts added to diets for turkeys on growth performance, gastrointestinal tract metabolism and carcass characteristics

A study on male turkeys was conducted to evaluate the physiological and growth effects of organic acids, organic acids with essential oils or herbal extract added to diets. The control diet (C) contained premix without any feed additives. Three experimental diets were fed from 1 to 140 days of age: diet A with a blend of organic acids (formic acid and propionic acid) in the amount of 5.0 g/kg of feed, diet AO with a blend of organic acids (citric, fumaric, orthophosphoric and malic acid) and with a mixture of essential oils (from citrus fruits, cinnamon, oregano and thyme) in the amount of 0.5 to 1.0 g/kg of feed, diet HE with preparation composed of oregano essential oil, spice extracts of turmeric and capsicum in the amount 0.3 g/kg of feed. Gastrointestinal tract development and metabolism (after 56 days of feeding) as well as growth performance and carcass characteristics (after 140 days of the experiment) were evaluated. Dietary supplementation with A, AO or HE signifi cantly decreased the pH of the crop contents, but had no effect on the pH of the caecal digesta. Dietary supplementation with A or HE increased the activity of α-glucosidase in the ileal digesta. The tested supplements had no infl uence on SCFA production in the caeca. All supplements increased the body weights of turkeys, but the differences were signifi cant (P≤0.01) only at the age of 84 days. The fi nal body weight (at 140 days of age) of turkeys fed diets with the tested supplements were higher by 3.3-3.9%, and feed conversion ratio decreased by 2.4-5.3% (statistically non-signifi cantly) in these treatments, compared to the control. Generally, carcass traits were not infl uenced by the above feed supplements.


INTRODUCTION
Organic acids, known to have strong antibacterial eff ects, are used for feed protection against microbes and fungi (Izat et al., 1990;Chaveerach et al., 2004;Immerseel et al., 2005).Acidifi ers are used as salmonella-control agents in feed and water supplied to livestock and poultry.Th e addition of organic acids to animal feed has been reported to decrease the intraluminal concentration of coliform bacteria and other acid-intolerant organisms, such as Campylobacter and Salmonella, known to be involved in digestive disorders (Canibe et al., 2001).A propionic acidbased product at 1.25% was quite eff ective in alleviating turkey poult enteritis and mortality syndrome (Roy et al., 2002).
However, professional literature indicates that the in vivo effects of different organic acids on intestinal bacteria vary a lot and depend on their chemical form and type of microorganisms, animal species, site and location in the gastrointestinal tract and buffering capacity of the feed.The effect of organic acids on broiler performance is dependent upon the dose and type of acid products, as well as on the fact whether the acids are included in feed or drinking water (Patten and Waldroup, 1988).
For many years, herbs and spices and their essential oils have been used as pharmaceuticals in natural therapy.In recent time, aromatic plants and their extracts have been used in animal feeding.Active substances naturally occurring in different plants, particularly in herbs used in human nutrition, have received increasing attention as possible AGP replacements (Nevas et al., 2004).Herbs, parts of herb plants or herbal plant extracts can benefi cially affect feed intake, secretion of digestive juices and the immune system of animals.
It is known that most of their properties are due to the essential oils they contain as products of their secondary metabolism (Adam et al., 1998).The essential oil derived from thyme and oregano (carvacrol and thymol) is known to possess in vitro antimicrobial, antifungal and antioxidant activities (Faleiro et al., 2005;Hazzit et al., 2006).Recent studies on experimental animals have revealed that some ingredients of popular herbs and spice plants, e.g., capsaicin or curcumin, can also stimulate digestive enzymes, bile fl ow and enhance bile acid secretion (Platel and Srinivasan, 2001).
Due to its in vitro antimicrobial activity, oregano essential oil may be expected to have prophylactic and therapeutic effects.Thus, oregano herb or essential oil or sole constituents of oregano essential oil have been tested as growth promoters in chickens and turkeys (Jamroz et al., 2003;Lee et al., 2003;Bampidis et al., 2005;Westendarp et al., 2006).However, the benefi ts of oregano for animal performance have not been consistent among these studies.
Only scant information is available on commercial herb extracts, bioactive essential oils or organic acids as feed additives in turkey diet.Therefore, the aim of this study was to examine the effect of wheat/maize-based diet supplementation with organic acids or natural plant extract on the gastrointestinal tract development, metabolism, performance and carcass characteristic of growing turkeys.Insomuch as the main action of feed additives is associated with the large bowel, special attention was paid to intestinal and caecal parameters (e.g., the activity of microbial enzymes and SCFAs).

Animals and diets
The experiment was conducted according to the guidelines of the Local Animal Experimentation Ethics Committee.
A total of 400 one-day-old heavy Large White BIG-6 turkey males, sexed at the local commercial hatchery were randomly assigned to four dietary treatments, each of four replications.The initial body weight of day-old poults was 61 g.Turkeys had free access to a diet without any of feed antibiotics but Clinacox TM (Diclazuril) as coccidiostat was used to 84 days of age.
The control diet (C) contained premix without any feed additives (Table 1).Three experimental diets contained commercially available products: a blend of organic acids (treatment A), a preparation with organic acids and essential oils (treatment AO) or preparation of herbal extracts (treatment HE).The A preparation containing a blend of formic acid and propionic acid (2.3:1) were applied in the amount 5.0 g/kg of feed.The AO preparation, containing a blend of citric acid (12%), fumaric acid (10%), orthophosphoric acid (6%) and malic acid (5%) with a mixture (2%) of hydrogenated essential oils from citrus fruits, cinnamon, oregano and thyme, was applied at the following doses: 1.0 g/kg (to 28 days of age), 0.75 g/kg (from 29 to 84 days of age) and 0.5 g/kg of feed (from 85 day).Diet HE contained a blend of oregano (Origanum vulgare L.) essential oil and spice extracts of turmeric (Curcuma longa L.) and capsicum (Capsicum frutenscens L.), were applied in the amount 0.3 g/kg of feed.
All feed mixtures were formulated using least-cost linear programming software to meet the nutrient requirements for turkeys, according to British United Turkeys (B.U.T., 2002) recommendation.The composition and calculated nutritive value of basal mixtures are given in Table 1.Starter mixtures were offered as crumbles and the other diets as 3 mm pellets.Fresh feed and water were provided daily and were available ad libitum.The ingredients of mixtures were mixed using a horizontal ribbon blade mixer with a capacity of 1,000 kg.Each batch was mixed for a period of 6 min, next put into a conditioner where steam was added at a pressure of 1.5 bar, and heated at a set temperature of 65°C for 10-15 sec.The temperature was monitored at the conditioner outlet and controlled by a Datastor computerized press controller (with a maximum variation from the set temperature of ±1°C).Pelletized diets were passed through a cooler.Pelleted feed was supplied by the local commercial animal feed mill.

Animals managament
All managament practices and euthanasia were carried out with full consideration of animal welfare.Poults were vaccinated for turkey rhinotracheitis (TRT) by spray application at 1 d of age and were allocated at random to 16 pens (25 turkeys per pen, 100 birds per treatment) with straw as bedding material.Stocking density was approximately 50 kg BW/m 2 of usable fl oor space in all pens.Visual health inspection of all birds was performed on a daily basis.Brooder rings for poults (till 10 days of age) and additional heat sources (till 28 days of age) were installed in the pens.Heating was provided by a central heating system and electric heaters (red light).The brooder unit's temperature was set at 35°C and then altered as needed to suit bird comfort.Room temperature was set at 28°C on the day of placement, and was subsequently reduced by 2°C per week.The temperature and humidity were recorded on a daily basis at 8 AM and 3 PM.The lighting program in the room was as follows: 23 h light at about 100 lux till 3 days of age and 14 h light at 5-6 lx from day 4 until the end of the experiment.Relative humidity was about 65 to 70%.Air changes were 0.4 -0.5 m 3 /h/kg of BW from 2 to 7 weeks of age and 0.7-0.8m 3 /h/kg of body weight from 8 weeks of age.

Basic experimental data
Mortality and the body weights of dead birds were recorded every day.Feed intake per replicate and the body weights of turkeys were recorded on day 28, 56, 84, 112 and 140.Gastrointestinal tract development and metabolism were evaluated after 56 days of feeding.Additionally, carcass characteristics (dressing percentage, abdominal fat and breast, thigh and drumstick muscles) were determined after 140 days of the experiment.

Gastrointestinal tract properties
After 56 days of experimental feeding, the birds were weighed and 10 turkeys representing the average body weight of each treatment were killed according to the recommendations for euthanasia of experimental animals (Close et al., 1997).Selected parts of the digestive tract (crop, gizzard, ileum, caeca) were removed and weighed.The pH of these sections was measured with a microelectrode and a pH/ ION meter (Model 301, Hanna Instruments, Vila do Conole, Portugal).Samples of the ileal (from the middle part of the ileum) and caecal contents were weighed and transferred to microfuge tubes, which were immediately stored at -40°C to measure dry matter content, digesta viscosity, short chain fatty acid (SCFA) content and the activity of microbial enzymes.The cecum part was fl ushed clean with ice-cold saline, blotted on fi lter paper, and weighed (caecal tissue weight).The same procedure was repeated for the small intestine.Dry matter of the ileal and caecal digesta was determined at 105°C.
Viscosity measurements of the ileal digesta were determined using a Brookfi eld cano-plate viscometer (Model LVDV-II+CP40, Brookfi eld Engineering Lab. Inc., Stoughton, MA).Samples of digesta (about 2 g from the middle part of the ileum between Meckel's diverticulum and ileocaecal junction) were centrifuged (13,000 rpm, 15 min at 20 o C) and the supernatant was collected.The viscosity of the supernatant was determined at 37 o C and with shear rates of 300-600 sec -1 .The reading was taken after one min.

SCFA concentration and activity of microbial enzymes
Caecal digesta samples were measured for SCFA concentration by gas chromatography (Shimadzu GC 14A, Shimadzu Co., Kyoto, Japan).Th e samples (of about 0.2 g) were weighed, mixed with 0.2 ml of formic acid, diluted with deionised water, and centrifuged at 10,000 g for 5 min.Th e supernatant was loaded onto a chromatography glass column, 2.5 m × 2.6 mm, containing commercial packing: 10% SP-1200/1% H 3 PO 4 on 80/100 Chromosorb W AW (Supelco Co., Bellefonte, PA).Th e chromatograph was coupled to a fl ame ionization detector, and we used a column temperature of 110°C, detector temperature of 180°C, and injector temperature of 195°C.
Microbial enzyme activity in the ileal and caecal digesta was measured by the rate of p-or o-nitrophenol release from their nitrophenylglucosides according to the modifi ed method of Djouzi and Andrieux (1997) described by Juśkiewicz et al. (2002).The following substrates (Sigma Chemical Co., St. Louis, MO) were used: for ß-glucuronidase, p-nitrophenyl-ß-D-glucuronide; for α-galactosidase, p-nitrophenyl-α-D-galactopyranoside; for ß-galactosidase, p-nitrophenyl-ß-Dgalactopyranoside; for α-glucosidase, p-nitrophenyl-α-D-glucopyranoside; and for ß-glucosidase, p-nitrophenyl-ß-D-glucopyranoside.The reaction mixture contained 0.3 ml of a substrate solution (5 mM) and 0.2 ml of a 1:10 (vol: vol) dilution of the digesta sample in 100 mM phosphate buffer (pH 7.0) after centrifugation at 10,000 g for 15 min.Incubation was carried out at 37°C, and p-nitrophenol was quantifi ed at 400 nm and o-nitrophenol at 420 nm after the addition of 2.5 ml of 0.25 M cold sodium carbonate.The enzymatic activity was expressed as micromoles of product formed per min (IU) per gram of digesta in the ileal and cecal sample.

Statistical analysis
All data were analysed statistically by a one-way analysis using a general linear model procedure for ANOVA and Duncan's multiple range test.Replicate means served as experimental units for statistical analysis.Treatment effects were considered to be signifi cant at P≤0.05 and P≤0.01.MIKULSKI D. ET AL.

RESULTS
Th e eff ects of feed additives on the performance of turkeys are presented in Table 2. Cumulative viability averaged 94-95% over the entire experimental period, and was not infl uenced by the tested feed additives.Th e applied supplements did not have a clear positive eff ect on performance of turkeys.At 28 and 56 days of age, the body weight of turkeys fed diets with A, AO or HE were higher by 1.9-3.7%than in the control group, but the eff ect was statistically non-signifi cant.At 84 days of age, the feed additives signifi cantly (5.6-6.6%;P≤0.01) improved the body weight of birds.At the end of the experimental period (140 days of age), the body weight of these turkeys were higher by 3.3-3.9%than in the control treatment, but this diff erence was statistically non-signifi cant.In comparison with the control group, the feed conversion ratio in the remaining treatments was lower by 1.3-4.0%(to 56 days of age) and by 2.4-5.3%(to 140 days of age), but these diff erences were statistically non-signifi cant.Th e use of feed additives had no signifi cant eff ect on the slaughter performance (Table 3).Only the proportion of thigh muscles was lower (P≤0.05) in the control turkeys than in those of treatment A.   Th e eff ects of organic acids (A), organic acids with essential oils (AO) or herbal extract (HE) on the functioning of the upper part of GIT of turkeys are presented in Table 4. Compared to the control treatment (C), diet supplementation with A and HE signifi cantly (P≤0.05) and with AO numerically decreased the pH of the crop contents.Th e pH of the ileal digesta in treatment HE was signifi cantly(P≤0.05)lower than in the control group.The ileal tissue weight in turkeys fed diets with AO or HE was by 10% (P≤0.05)higher than in treatment A. The weight of the ileal digesta of turkeys of the control group (C) was signifi cantly higher, compared to all other treatments (13.1 vs 6.2-8.9 g/kg of BW).It corresponded with higher viscosity in the control group than in turkeys fed diets with A, AO or HE supplementation.Dry matter concentration in the ileal digesta were unaffected by the treatments.
Microbial glycolytic enzyme activity in the ileal digesta was relatively low.Compared to the control treatment, dietary supplementation with A or HE signifi cantly increased (P≤0.05) the activity of α-glucosidase in the ileal digesta.
The caecal tissue weight and the weight of the caecal digesta in turkeys (Table 5) fed diets with A, AO or HE supplementation were numerically higher than in the control treatment (C), but the effect was statistically non-signifi cant.On the other hand, the DM content of the digesta in these treatments was by 3.1-10.4%lower than in the control group (no statistically signifi cant differences).The addition of A, AO or HE had no effect on the pH of the caecal digesta.
In the caeca, the highest activity of α-glucosidase and α-galactosidase was recorded in treatments A or AO.Compared with treatment HE, signifi cantly (P≤0.05)higher activity of β-glucosidase in treatment A, as well as higher activity of β-galactosidase in treatments A and AO, were found.The dietary supplementation of formic and propionic acid (treatment A) signifi cantly increased (P≤0.05) the activity of potentially detrimental β-glucuronidase in the caeca.
After 56 days of experimental feeding, no infl uence of feed additives on the total production of short-chain fatty acids in the caecal digesta (SCFA pool) was observed.Signifi cant differences (P≤0.05) were noted in valerate acid concentration only in the case of treatments C and AO, but this acid does not make up the essential part of the SCFA pool.At the same time, the proportions of major fatty acids (acetate, propionate and butyrate) were similar.Statistically nonsignifi cant increase in butyrate acid proportion, as compared to the proportion of acetic acid and propionic acid, was observed in turkeys fed diets with AO.

DISCUSSION
Like antibiotics, short-chain organic acids have a specifi c antimicrobial activity, particularly effective against acid-intolerant species (E.coli, Salmonella and Campylobacter).Organic acids have also several additional effects.These include reduction in digesta pH, increased pancreatic secretion, and trophic effects on the gastrointestinal mucosa.Organic acids have a clear and signifi cant benefi t in weanling piglets.The data on the use of organic acids or natural feed additives as alternatives to antibiotic growth promoters in poultry diets (mainly in diets for broilers and turkeys), published to date, are, however, inconsistent.Lack of consistency in demonstrating an organic acid benefi t is related to uncontrolled variables such as buffering capacity of dietary ingredients, presence of other antimicrobial compounds, cleanliness of the production environment, and heterogeneity of gut microbiota.A reduction in caecal pH of broiler chickens was observed when a blend of formic acid/propionic acid was added at 1.0% of feed (Waldroup et al., 1995).However in other studies the application 5 and 10 g/kg feed of formic acid (Hernandez et al., 2006) or buffered propionic acid (Izat et al., 1990) had no effect on intestinal pH in broiler chickens.Similarly, acidifi ed water containing a commercial organic acid product at pH 4.0 had no signifi cant infl uence on the pH and SCFA concentrations in the crop and caecal contents of broilers (Chaveerach et al., 2004).The inclusion of formic and propionic acids to the feed of hens made no difference to the pH of the intestinal Essential oil rich in carvacrol (applied alone or in combinations) is known to have a growth-stimulating effect in piglets (Manzanillo et al., 2001) or broiler chickens (Jamroz et al., 2003).In contrast, many authors reported no infl uence of carvacrol, thymol, oregano essential oil or commercial products with oregano or dried oregano leaves on the performance of broiler chickens and turkeys (Lee et al., 2003;Bampidis et al., 2005;Westendarp et al., 2006).In the present study the body weight of turkeys fed diet with carvacrol-based essential oil (HE treatment) increased, but only to 84 days of age.After 140 days of feeding, the fi nal body weight of turkeys were only slightly higher (P>0.05) and feed conversion ratio was numerically lower (P>0.05) in this treatment than in birds fed a non-supplemented diet.The lack of a growth-promoting effect of herbal mixture supplements in these studies may be related to diet composition and environmental conditions.No effect of oregano on growth performance is seen when feeding a well-balanced diet and keeping the birds in a clean environment (Lee et al., 2003), but this does not rule out the possibility that positive effects would have been observed under worse hygienic environmental conditions or when using a less digestible diet.
Our results and the fi ndings of other authors (Bampidis et al., 2005;Westendarp et al., 2006) show that the carcass traits were not infl uenced by feed supplements containing herbs or essential oils.In our study the only exception was the proportion of thigh muscles in birds from treatment A. However, in an experiment by Izat et al. (1990) dietary supplementation with buff ered propionic acid significantly increased the carcass dressing percentage of chickens.

CONCLUSIONS
In conclusion, the results of our study indicate that when using turkeys under conditions of good hygiene and fed optimal diet, dietary supplementation with organic acids, essential oils or a combination of organic acids and essential oils did not have a clear positive eff ect on performance and carcass traits, however, there was a slight positive eff ect on gastrointestinal tract metabolism.Th e tested supplements caused a decrease in the pH of the crop digesta and in ileal digesta viscosity as well as an increase in the activity of bacterial enzymes in the caeca, but had no eff ect on SCFA production.All supplements increased the body weight of turkeys, but only to 84 days of age.Th e fi nal body weights (at 140 days of age) of turkeys fed diets with the tested supplements were higher by 3.3-3.9%,and feed conversion ratio decreased by 2.4-5.3%(statistically non-signifi cantly) in these treatments, compared to the control.
means within the same line with no common superscripts differ (P≤0.01) 1 data represent mean values of four replicate pens of 25 turkeys each 2 experimental diets: C = basal mixture without any feed additives; A = basal mixture + organic acids; AO = basal mixture + preparation with organic acids and essential oils; HE = basal mixture + preparation with oregano essential oil and spice extracts of turmeric and capsicum

Table 2 .
Effect of diets containing organic acids or herbal extract on the body weight (BW), feed conversion ratio (FCR) and mortality of turkeys raised from 1 to 140 days of age 1

Table 3 .
Effect of diets containing organic acids or herbal extract on the carcass characteristic of turkeys at the age of 140 days, % of body weight 1

Table 4 .
Effect of diets containing organic acids or herbal extract on the functioning of the upper part of gastrointestinal tract of turkeys at the age of 56 days 1 -b means within the same line with no common superscripts differ (P≤0.05) 1 data represent mean values of 10 turkeys per treatment 2 as in Table 2 MIKULSKI D. ET AL. a

Table 5 .
Effect of diets containing organic acids or herbal extract on the functioning of the caeca of turkeys at the age of 56 days 1