Effects of the Essential Oil Blend CRINA^® Poultry in Feed on Broiler Performance and Gut Microbiology

Abstract

The aim of the present study was to investigate the influence of a commercial essential oil blend (EO), CRINA^® Poultry (CRP), as an alternative to in-feed antibiotic, enramycine^®, on growth parameters of broilers during pre and post-challenge with Clostridium perfringens (C. perfringens). The birds received treatments from 0 to 30 days as follows: T1, positive control (+CONT): without medication or bacterial challenge; T2, negative control (-CONT): without medication but with C. perfringens challenge; T3, antibiotic with addition of enramycin with C. perfringens challenge (ENRA); and T4, with addition of CRINA^® Poultry to feed with C. perfringens challenge (CRP). Overall, feed intake (FI), body weight gain (BWG) and feed conversion ratio (FCR) was not significantly different among the four treatments (P>0.05). On the other hand, EO supplementation caused some changes in the gut morphology, birds which had received CRP had longer ileal villi as compared to +CONT or ENRA groups (P<0.05). The performance of the birds which had consumed CRP was similar to all other treatments. On the other hand, the results obtained from this trail clearly showed that CRP supplementation failed to control the proliferation of C. perfringens in the intestine. The results from this study indicated that CRP under the condition of this experiment had no influence on broilers performance but it had a positive effect on gut morphology.

KeyWords:

• Broiler
• Crina^®Poultry
• Enramycine
• Intestinal mucosa morphometrics
• Performance

Introduction

The advantages of antibiotic as antimicrobial growth promoters (AGPs) for poultry when used at subtherapeutic levels are well documented (Hume et al., 2011). The AGPs have been used in animal nutrition since 1946, when their positive effects were observed for the first time (Moore et al., 1946). However, recently there are increasing concerns about the risk of developing cross-resistance and multiple antibiotic resistances in pathogenic bacteria, which could result in proliferation of antibiotics-insensitive bacteria; this could lead to a decrease in the therapeutic effectiveness of antibiotics used by humans (Castanon, 2007). Current trends in poultry production point to reduce the use of AGPs and increase the use of non-antibiotic feed additives. Essential oils (EO) are among the alternative growth promoters that are already in use in practice (Mitsch et al., 2004).

Essential oils are extracted from herbs and spices by steam distillation, most of these compounds are phenolic (thymol (thymus vulgaris), eugenol (Syzygium aromaticum), curcumin (Curcuma peppermint) and piperin (Piper nigrum) with varying antimicrobial activity (Lee et al., 2004). Several reports showed that specific components of EO inhibit growth of many bacteria, including C. perfringens in vitro (Briozzo et al., 1988; Dorman and Deans, 2000) and in vivo (Mitsch et al., 2004). On the other hand, dietary concentrations of 100 and 200 mg/kg feed of CRINA^® Poultry (CRP) failed to reduce the intestinal numbers of C. perfringens compared to a control group (Abildgaard et al., 2010)

The effect of EO on broilers performance is controversial and depends on the active material of the EO. Tiihonen et al., (2010) reported that a blend of EO (thymol and cinnamaldehyde) added to SBM-wheat-based diet increased body weight gain (BWG) of broilers from 0 to 42 d by 45%. Likewise, Francesch et al., (1999) noted that CRINA^® Poultry supplementation to wheat and-barley-based diet improved feed conversion ratio (FCR) in broilers. On the other hand, the supplementation of CRP blend to coccidia vaccinated broilers did not show any benefits in older birds (Oviedo-Rondón et al., 2006). Likewise, CRP failed to improve BWG and FCR of broilers which were vaccinated against coccidiosis (Abildgaard et al., 2010). Further, Shanmugavelu et al. (2004) concluded that EO from thyme and garlic did not improve the nutritional value of the diet.

Therefore, this study was conducted to investigate the efficacy of a commercial EO (CRINA^® Poultry) to replace typical AGPs (enramycine) and compare their effects on growth parameters, histomorphology and ileal bacterial count of broilers raised in cages under C. perfringens challenge.

Materials and methods

Animals, husbandry and treatments

A total of one hundred, 0-day-old male broiler chicks (Ross 308) were obtained from a commercial hatchery and vaccinated for Marek’s disease, Newcastle and infectious bronchitis. At arrival, chicks were sexed, grouped by weight to reduce variation in mean body weight, and then were allotted to 20 cages according to the treatment. Five chicks were placed in each cage (50 cm length, 60 cm width and 36 cm depth) in a four-deck cage system and received the experimental diets in electrically heated battery brooders with raised wire floors.

Birds received one of the following four treatments: T1, control diet (+CONT): without medication or bacterial challenge; T2, T1 + C. perfringens challenge (-CONT): without medication but with bacterial challenge; T3, 0.1 g/kg of feed of enramycin (ENRA) with bacterial challenge; T4, 0.01% CRINA^® Poultry (CRP) with bacterial challenge. The level of CRP was based on the manufacturer recommendations. The EO component used in this study is (CRINA^® Poultry, DSM Nutritional Products Ltd., Basel, Switzerland), is commercially available and contains primarily 29% of active components, including thymol, according to the manufacturer. On Day 16, birds in treatments two to four were challenged with C. perfringens using 10-fold dose of anticoccidial vaccine (Paracox-8), on Days 18 and 20 chicks were gavaged with 1 mL of a cocktail containing C. perfringens inoculations (4x10⁸ CFU). Culture of C. perfringens was obtained commercially (MicroBiologics, Cloud, MN, USA).

A typical isocaloric and isonitrogenous starter (0-16 d) and finisher (17-30 d) diets based on corn-soybean meal diets were formulated in mashed form which met or exceeded the recommendations in commercial practice in Saudi Arabia for Ross (Table 1). Ambient temperature and relative humidity were concurrently and continuously recorded at 3 hours interval using two data loggers (HOBO Pro Series Data Logger, Model H08-032-08, Onset Co., Bourne, MA, USA) placed inside the chamber. The average temperature and relative humidity for the whole period were 24.95°C±0.26 (SD) and 26.63%±3.30 (SD), respectively. The study was conducted under a protocol approved by King Saud University and complies with the current laws of Saudi Arabia.

Measurements

Feed intake and BWG were recorded weekly by pen and FCR computed at 16 and 30 day of age. Mortality was checked daily and weights of dead birds were used to adjust FCR. At the end of the trail (30 days), five birds per treatment were selected, after euthanasia, feather and skin, heads, necks, and shanks were removed, and the remaining carcasses were dissected to breast and leg quarter and were weighed. The percentage of yield of each part was calculated on the basis of dressed weight. At 16 and 30 days of age, the entire small intestine tract of all birds was removed aseptically, and then weight and length of different parts of intestine including, duodenum, jejunum and ileum were taken. A 2-cm-long sample from each portion of the small intestine was collected for histology measurements (Peinado et al., 2012). Samples were fixed in phosphate-buffered formalin for at least 48 h, after which they were embedded in paraffin. Sections of 5 mm were cut and stained with haematoxylin and eosin for measurements of height and width which were based on at least 4 well-oriented villi per section per broiler. The measurements were done by using an IX71 Inverted Olympus Microscope (Eyepiece: WH10X, Objective Lens: 4X). Cellsens Digital Imaging Software for Research Application software was used for calculations.

Ileal digesta contents were aseptically emptied in a new sterile bag and kept in ice until time of analyses. Samples were diluted in 0.9% saline and 0.1 mç of each sample was plated on duplicates by using selective agar media for enumeration. Tryptose sulfite-cycloserine (TSC) agar media was used for C. perfringens enumeration (Oxoid CM587 with the addition of SR88 and SR47). Colonies on TSC agar that were suspected to be C. perfringens were plated secondarily on blood agar (Garridol et al., 2004). Enterobacteriaceae and Salmonella were identified by API 20E. The API 20E strips (bioMérieux, Craponne, France) were inoculated, incubated at 37°C for 24 h and interpreted as recommended by the manufacturer. Reactions were recorded and identifications were determined by using a computer program [API Lab Plus software version 3.2.2 (bioMérieux)]. Results were expressed as log₁₀ colony-forming units per gram of ileal digesta (log CFU/g).

Table 1. Composition of the experimental diets.

	Starter			Finisher
1 & 2	3^°	4^°	1 & 2	3^°	4^°
Ingredients, %
Yellow corn	56.00	55.99	55.99	57.75	57.74	64.12
Soybean meal	36.10	36.10	36.10	34.0	34.0	34.0
Palm oil	3.80	3.80	3.80	4.80	4.80	4.80
DCRP	2.30	2.30	2.3	2.0	2.0	2.0
Ground limestone	0.72	0.72	0.72	0.64	0.64	0.64
Choline chloride	0.10	0.10	0.10	0.05	0.05	0.05
DL-methionine	0.23	0.23	0.23	0.16	0.16	0.16
L-lysine	0.15	0.15	0.15	-	-	-
Salt	0.30	0.30	0.30	0.30	0.30	0.30
Vitamin premix^#	0.25	0.25	0.25	0.25	0.25	0.25
Trace mineral mix^§	0.05	0.05	0.05	0.05	0.05	0.05
Enramycin	-	0.01	-	0.00	0.01	0.00
CRINA Poultry	-	-	0.01	-	-	0.01
Total	100	100	100	100	100	100
Calculated analysis
Metabolizable energy, kcal/kg	3000	3000	3000	3100	3100	3100
Crude protein, %	22.0	22.0	22.0	21.0	21.0	21.0
Non phytate P, %	0.45	0.45	0.45	0.40	0.40	0.40
Calcium, %	1.0	1.0	1.0	0.9	0.9	0.9
Lysine, %	1.25	1.25	1.25	1.1	1.1	1.1
Methionine, %	0.55	0.55	0.55	0.47	0.47	0.47

°Diet 3 had 0.01% enramycin, diet 4 had 0.01% CRINA Poultry on the expense of corn during starter and finisher.

^#Vitamin mix is supplied in the following per kg of diet: retinyl acetate, 3.41 mg; cholecalciferol, 0.07 mg; DL-α-tocopheryl acetate, 27.5 mg; menadione sodium bisulphate, 6 mg; riboflavin, 7.7 mg; niacin, 44 mg; pantothenic acid, 11 mg, cyanocobalamin, 0.02; choline 496 mg; folic acid, 1.32 mg; pyridoxine HCl, 4.82 mg; thiamine mononitrate, 2.16 mg; D-biotin, 0.11 mg.

^§Mineral-mix is supplied in the following per kg of diet: manganese, 67 mg; zinc, 54 mg; copper, 2 mg; iodine, 0.5 mg; iron, 75 mg; selenium, 0.2 mg.

Table 2. Body weight gain, feed intake and feed conversion ratio of broiler chickens given experimental diets at different ages.

	Treatment				SEM	P
	T1	T2	T3	T4
Performance at 16 d
BWG, g	479.2	481.2	515.2	486.2	±14.0	ns
FI, g	638.0	645.6	652.6	631.0	±20.8	ns
FCR, g:g	1.333	1.342	1.267	1.300	±0.03	ns
Performance at 30 d
BWG, g	886.3	832.1	869.5	880.4	±26.7	ns
FI, g	1489.6	1468.9	1432.5	1464.6	±36.4	ns
FCR, g:g	1.718	1.770	1.656	1.666	±0.04	ns
Cumulative
BWG, g	1345.3	1313.3	1384.6	1366.5	±34.5	ns
FI, g	2127.4	2114.5	2084.8	2095.8	±36.1	ns
FCR, g:g	1.583	1.611	1.511	1.535	±0.03	ns
Livability, %	100.0	92.0	92.0	92.0	±6.939	ns

BWG, body weight gain; FI, feed intake; FCR, feed conversion ratio. ns, not significant.

Table 3. Effect of different treatments on relative weight of different body sections at 30 days of age.

	Treatment				SEM	P
	T1	T2	T3	T4
Dressed yield, %	60.9	60.7	62.1	58.9	±1.38	ns
Breast^°, %	35.6	35.2	34.2	34.3	±0.56	ns
Leg quarter^°, %	40.4	40.3	40.2	41.2	±0.61	ns
Abdominal fat, %	1.05	0.80	1.26	1.14	±0.20	ns
Liver, g/100 g	0.31	0.24	0.28	0.31	±0.02	ns

°Breast and leg quarter were expressed as percentage of the carcass weight. ns, not significant.

Table 4. Intestinal morphology and histology of broilers at 16 days of age.

	Treatment	SEM	P
	T1	T2	T3	T4
Intestine length, cm	129.7	132.0	127.0	130.3	±7.20	ns
Duodenum length, %	19.5^a	17.4^b	16.8^bc	15.9^c	±0.44	^**
Jejunum length, %	47.8^a	41.6^b	41.5^b	39.9^b	±0.71	^***
Ileum length, %	32.7^c	41.1^b	41.7^ab	44.3^a	±0.86	^***
Intestine weight, g/cm	0.28	0.21	0.27	0.24	±0.02	ns
IRW, g/100g BW	7.4	7.6	7.2	8.0	±0.74	ns
Ileal villus height^°, µm	4509^a	4296^ab	380^4b	4378^a	±155.7	^*
Ileal villus width^°;, µm	693	766	653	879	±59.9	ns

IRW, intestine relative weight; BW, body weight.

°Measurements of height and width were based on at least 5 well-oriented villi per ileum per broiler for a total of 5 birds per treatment.

^a,b,cMeans in the row with different superscripts differ significantly (*P<0.05; **P<0.01; ***P<0.001). ns, not significant.

Table 5. Intestinal morphology and histology of broilers at 30 days of age.

	Treatment				SEM	P
	T1	T2	T3	T4
Intestine length, cm	152.0	178.0	176.2	174.3	±6.60	ns
Duodenum length, %	15.7^ab	16.6^a	14.8^bc	16.6^a	±0.37	^*
Jejunum length, %	52.1	41.8	43.2	41.7	±4.18	ns
Ileum length, %	32.2	41.7	42.1	41.7	±4.19	ns
Intestine weight, g/cm	0.36	0.44	0.49	0.49	±0.03	ns
IRW, g/100g BW	4.7	5.7	5.5	6.4	±0.35	ns
Villus height^°, µm
Duodenum	8964	8315	8480	10068	±445	ns
Jejunum	7208	8449	9467	9659	±597	ns
Ileum	5869^bc	6643^ab	4692^c	7664^a	±515	^*
Villus width^°, µm
Duodenum	1245^b	1298^b	1647^a	1316^b	±139	^*
Jejunum	1109	938	1088	1245	±81	ns
Ileum	843	882	851	736	±108	ns

IRW, intestine relative weight; BW, body weight.

°Measurements of height and width were based on at least 5 well-oriented villi per ileum per broiler for a total of 5 birds per treatment.

^abcMeans in the row with different superscripts differ significantly (*P<0.05). ns, not significant.

Table 6. Ileal bacterial count in broilers at 16 and 30 days of age (mean, log₁₀ CFU/g).

	Treatment				SEM	P
	T1	T2	T3	T4
Starter
C. Perfringens	4.2	3.9	4.3	4.8	±0.20	ns
Gram negative bacilli	3.8	4.3	4.3	3.8	±0.28	ns
Finisher					ns
C. Perfringens	4.7	5.8	4.8	3.4	±0.91	ns
Gram negative bacilli	4.8	4.7	5.0	5.0	±0.18	ns

Statistical analysis

Data were analyzed by using the general linear model procedure of SAS (2002). Four treatments were replicated five times in a randomized complete block design, a cage constituted the experimental unit. Means for measurements showing significant differences in the analysis of variance were tested using the PDIFF option. Means ± SEM are presented in the tables and differences were considered statistically significant at P<0.05.

Results and discussion

Feed intake, BWG and FCR of broilers at different ages are shown in Table 2. During the starter (0-16 day) and the finisher (17-30 day) periods, BWG, FI and FCR were not influenced (P>0.05) by treatment. As a result, cumulative (0-30 day) FCR was not significant (P>0.05), even though there was a numeric improvement in cumulative FCR for birds which had received ENRA or CRP as compared to -CONT (1.511, 1.535 and 1.611 g:g, respectively). The mean percentage of carcass parts in different treatments is documented in Table 3. Treatment had no effect on dressing percentage of breast muscle yield, leg quarter yield, abdominal fat and relative liver weight (P>0.05). The results of this study are in general agreement with previous reports (Abildgaard et al., 2010; Oviedo-Rondón et al., 2006). It is worth mentioning that both previous reports used CRP as EO and the birds were vaccinated against coccidiosis. However, in the current study, a mega dose of anticoccidial vaccine (Paracox-8^®, Schering-Plough Animal Health, Uxbridge, UK) was used as a part of the protocol to induce the C. perfringens challenge. The inclusion level of CRP recommended by the manufacturer was used in this trial, so the lack of effect of the supplements on performance could be related to the anticoccidial vaccine. Other reports showed that dietary essential oil components improve performance of broiler chickens through different mechanisms such as stimulating the activity of endogenous digestive enzymes and enhancing nutrient digestibility. (Francesch et al., 1999; Tiihonen et al., 2010; Williams and Losa, 2001).

The morphometric measurements of the intestinal epithelium samples at 16 days of age are given in Table 4. Percentage of duodenal, jejunal and ileal length showed significant differences due to treatments (P<0.01, 0.001, 0.001, respectively). Duodenum and jejunum from birds which had received +CONT were the longest among all treatments while the ileum from the same group was the shortest. Intestine weight and relative weight were not affected by any treatment (P>0.05). Ileal villus height was significantly shorter for birds which had received ENRA. On the other hand, ileal villus width was not influenced by treatment (P>0.05). At 30 days of age, duodenum length of birds treated with ERNA was shorter (P<0.05). Similar result was reported by Oliveira et al. (2008) who found that antibiotic supplementation caused low villi height; they explained that by the suppressing effect of the antibiotic for the beneficial bacteria in the gut, as Lactobacillus and bifidobacteria. On the other hand, birds which had received CRP had longer ileal villi (P<0.05); while those received ENRA had wider duodenal villi as compared to all other treatments (P<0.05). Long villi are usually equated with excellent gut health, high absorptive efficiency and healthier intestinal tract of chickens (Alfaro et al., 2007). According to Cera et al., (1988), maximum absorption and digestion capacity is provided by a large luminal area with villus height and mature enterocytes and is essential for animal growth. Jejunal villus height and width were similar among all treatments (P>0.05).

Data related to ileal bacterial count in broilers at 16 and 30 d of age are presented in Table 6. Similar bacterial count of C. perfringens and gram negative Bacilli were found in the starter (before the challenge) and finisher (after the challenge) periods (P>0.05). The results obtained from this trail clearly showed that CRP supplementation failed to control the proliferation of C. perfringens in the intestine. Similar conclusion was obtained by Abildgaard et al. (2010) by using two levels of CRP (recommended by the manufacturer and double the dose); in both cases the EO did not reduce the intestinal numbers of C. perfringens compared to a non-supplemented group. Several other groups used the same EO supplement as in this experiment and came to different findings. Losa and Kohler (2001) found a reduction in the C. perfringens count of the intestinal content by using 50 ppm of CRP. Similarly, the findings of Mitsch et al. (2004) correspond to those of Losa and Kohler (2001).

In this experiment, birds were challenged with C. perfringens to mimic a challenge that a bird could face in rearing facilities. According to Porter (1998) C. perfringens assumed to be the main health problem associated with removing the antibiotics from feed. Clostridium perfringens infection of broilers may cause impairment of production performance (Lovland and Kaldhusdal, 2001) by increasing activity of bile salt hydrolase which led to growth depression (Feighner et al. 1987). Also, C. perfringens can cause a subclinical disease associated with necrotic enteritis (NE) which is characterized by damage to the intestinal mucosa that decreases digestion, absorption and reduces weight gains. Ficken and Wages (1997), reported pathological changes in the intestinal tissue of birds challenged with C. perfringens. The gross examination of the responses in birds challenged orally with C. perfringens showed sub-clinical inflammatory responses throughout various sections of gizzard, duodenum, jejunum, ileum, and ceca associated with intestinal lesions and hemorrhages. However, none of the challenged birds produced overt clinical signs of necrotic enteritis and there were no mortalities associated with oral exposure to high doses of C. perfringens. They suggested this may be due to the absence of stress related to other diseases, diets or environment; all these factors with the presence of C. perfringens could cause an outbreak of necrotic enteritis.

Conclusions

In this experiment, CRP failed to reduce ileal bacterial count as a result the performance was not improved significantly. Essential oil supplementation caused some significant changes in the gut morphology; birds which had received CRP had longer ileal villi as compared to +CONT or ENRA groups that might be due to better absorption capacity.

Acknowledgments

The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group Project No. RGP-VPP-273.