Effects of a phytogenic feed additive on growth performance, selected blood criteria and jejunal morphology in broiler chickens

The study was conducted to examine the effects of a phytogenic feed additive (PFA; BIOSTRONG® 510) on growth performance, selected blood parameters and jejunal morphology in broiler chickens. The PFA consists of a mixture of essential oils with thymol and anethole as leading active substances, as well as different herbs and spices. A total of 264 1-d-old Cobb male broilers were randomly allocated to 2 dietary treatments, with 6 replicates per treatment and 22 birds each replicate. The experiment lasted 42 days. The dietary treatments were a starter and grower diet without feed additives (control), or the diets supplemented with 150 mg/kg of the PFA. Body weight and feed intake were not significantly influenced by PFA feeding compared with the control during all experimental periods. During the grower phase (22 42 d of age) and during the whole period (1 42 d of age) the PFA significantly improved (P<0.05) feed conversion ratio. Serum total protein, albumin, total cholesterol and leucocytes were increased by PFA feeding. Furthermore, villus height to crypt depth ratio in the jejunum was increased by PFA feeding. In conclusion, the results of this study show that inclusion of PFA lead to morphological changes in the jejunum, which might influence nutrient absorption and thus, improved FCR.


Introduction
The ban of antibiotic growth promoters (AGP) in poultry feeds intensified the search for alternatives improving the health and productivity of broiler chickens (Barreto et al., 2008).Such alternatives are probiotics, prebiotics and organic acids, which are added to the feed (Huyghebaert et al., 2011).Also phytogenic feed additives (PFA) were shown to enhance performance in AGP-free livestock production (Alçiçek et al., 2003;Steiner, 2009).Phytogenic feed additives consist of a broad variety of substances, mainly extracts from plant materials, such as flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits and roots (Burt, 2004).The active molecules include many different secondary plant metabolites, resulting in a broad range of physiological effects, like secretolytic and spasmolytic, or immune-stimulative effects (Lee et al., 2004a).
PFA are generally recognized as natural feed additives and safe to the animal.However, results of studies concerning the use of PFA in broiler nutrition are inconsistent (Windisch et al., 2008).Some authors stated significant improvement of broiler performance (Ertas et al., 2005;Cross et al., 2007), whereas others reported no effects on BW gain and feed intake (Lee et al., 2003;Jamroz et al., 2005;Nasir and Grashorn, 2010) or feed conversion ratio (Ocak et al., 2008).These discrepancies may be due to numerous factors such as type and parts of plants used, their physical properties, time of harvest, the preparation method of PFA and their compatibility with other feed components (Jang et al., 2007).Furthermore, the mode of action of these additives is not fully clarified yet and in vivo studies are limited.Plant extracts have been shown to influence digestion and secretion of digestive enzymes (Platel and Srinivasan, 2000;Williams and Losa, 2001) to increase absorption of micronutrients (Usha et al., 2010) and to exhibit antibacterial, antiviral and antioxidant activities (Brenes and Roura, 2010).However, only few studies investigated the effects of PFA on intestinal morphology in broiler chickens.Therefore, the aim of the present study was to examine the effect of a commercial PFA on performance, intestinal morphology and selected blood parameters in broiler chickens.

Materials and Methods
Two hundred sixty four 1-d-old male Cobb chickens were weighed and randomly allotted to 2 experimental treatments.Each treatment consisted of 6 replicates with 22 birds per replicate.The dietary treatments were a starter and grower basal diet without any feed additives used as a control or the basal diets supplemented with 150 mg/kg of a commercial PFA (BIOSTRONG ® 510, Delacon, Steyregg, Austria) used as experimental diets.The PFA consisted of a mixture of essential oils, with thymol and anethole as leading active substances, as well as different herbs and spices.The compositions of the basal diets are shown in Table 1.Control and experimental diets were formulated to be iso-nutritive and to meet the requirements for broiler chickens during starter and grower phase.Feed and water were available ad libitum.Feed consumption and BW of the birds were recorded weekly and were used to calculate broiler performance (weight gain, feed intake, feed conversion ratio).
Blood samples were taken at 35 d of age from the brachial vein from 12 birds per treatment group (2 birds per pen).Blood samples were separated by centrifuge, and serum was analyzed for glucose, total cholesterol, triglycerides, albumin and total protein using commercial test kits.Blood cell counts were determined by commercial cell counter.
At 42 d of age, 9 birds per treatment were slaughtered and the jejunum was removed for assessment of tissue morphology.The tissue samples were taken from the middle part of the jejunum (from the entrance of bile duct to Meckel's diverticulum, pieces about 5 cm in length), carefully cleansed and then fixed in 4% buffered formalin for 2 days.The further processing consisted of serial dehydration in PBS and graded ethanol solutions, clearing with xylene and embedding in paraffin.Sections of 5 µm were prepared and placed on glass slides.Tissue samples were deparaffinised, rehydrated and stained with haematoxylin and eosin.Villus heights and crypt depths were examined with a photomicroscope (Photomikroskop III, Carl Zeiss, Oberkochern, Germany) fitted with a digital camera (MikroCam 3 MP, Bresser, Rhede, Germany) and images were analysed using image analysis software (Image software Bresser MikroCamLab Mikroskopie).A total of 16 intact well-oriented villus-crypt units were randomly selected at each tissue sample.Villus height was measured from the tip of the villus to the villus-crypt junction, and crypt depth was defined as the depth of the invagination between two villi.
Experimental data were analyzed by ANOVA using SPSS v. 17.0 (Statistical Packages for the Social Sciences, released August 23, 2008).Data was tested for homogeneity of variances, and comparison of means was performed by Tukey test.The significance level was set at p <0.05.

Results
During the whole experiment, birds were healthy and the mortality was below 1%.Performance data for broiler chickens during the starter and grower phase and for the total experimental period are summarized in Table 2.For all experimental periods, there was no effect (P>0.05) of the PFA on BWG and feed intake.Feed conversion ratio was significantly improved (P<0.05) in the grower phase (22 -42 d of age) as well as during the whole experimental period (1-42 d of age) due to PFA supplementation.
Villus heights, crypt depths and villus height: crypt depth ratios of jejunal tissue samples are presented in Table 4.In broilers fed diets supplemented with PFA, there were no differences between the villus height and crypt depth could be observed compared to the control (Figure 1).Villus height: crypt depth ratio was significantly (P<0.05)increased in birds fed PFA compared to the control.
Effect of the phytogenic feed additive on some blood parameters in broilers were summarized in Table 3. Serum total protein, albumin, cholesterol and leucocytes were increased (P<0.05), while serum glucose, triglycerides, hemoglobin and erythrocytes were not affected by PFA feeding.

Discussion
In the present study, there was no effect of the tested PFA on BW and feed intake in broilers.These results are consistent with the studies of Jang et al. (2007) and Erdogan et al. (2010), who did not find effects of different phytogenic compounds on broiler growth performance.However, FCR was significantly improved in the present study.Consistently, Jamroz et al. (2005) reported improved FCR due to the addition of a plant extract, containing cinnamaldehyde, carvacrol and capsaicin, to a maize or wheat and barley based diet by 4.1 or 2.0%, respectively, whereas BW was not affected by treatment.Al-Kassie (2009) showed that the addition of 200 ppm oil extract derived from thyme and cinnamon to broiler diets significantly improved BW gain and FCR during a growing period of 6 weeks and Ocak et al. (2008) reported higher (P< 0.05) BW at 21 and 42 d of age as well as higher (P< 0.05) BW gain from 7 to 35 days of age in broilers fed peppermint and thyme compared to the controls.The above mentioned studies, reporting improved FCR, have in common, that the added substances mainly consist of essential oils.Essential oils exert antimicrobial activity in the digestive tract of animals (Lee et al., 2004;Al-Kassie, 2010).It is hypothesized that gut microflora reduces nutrients available to the animal by enforcing the intestinal cell turnover and thereby increasing the intestinal requirement for nutrients to maintain tissue integrity.Moreover, intestinal microflora and epithelial cells have to compete for nutrients (Dibner and Richards, 2005;Lan et al., 2005).By a reduction of intestinal microflora, essential oils may lead to moderate cell turnover, to decreased intestinal nutrient requirements and to less competition for available nutrients.As a consequence, FCR is improved because more nutrients are used for BW gain instead of tissue maintenance or microbial growth.
The increased nutrient supply for growth is reflected in enhanced nutrient transport in the blood.For example, Ghazalah and Ali (2008) observed higher levels of total protein, albumin and globulin in the blood serum of birds when fed 0.5% rosemary leaves.Similar results were obtained in the present study, where higher contents of total protein and albumin in the blood serum of PFA fed animals may also indicate enhanced nutrient supply and transport.Furthermore, Tekeli et al. (2006) reported increased blood glucose concentration by Zingiber officinale supplementation and increased triglyceride concentration by both Zingiber officinale and Syzygium Aromaticum supplementation.In the present study, there was no effect of PFA on blood glucose and triglyceride concentrations.Calislar et al. (2009) found effects neither on triglycerides nor on blood cholesterol levels by application of a PFA containing Origanum vulgare ssp.hirtum extract.In general, essential oils are more often associated with hypocholesterolemic properties (Lee et al., 2004).In contrast, in the present study chicken fed the PFA showed higher serum cholesterol concentrations compared to the control animals.This discrepancy may be due to the combination of essential oils and pungent substances which were also included in the used PFA.Pungent substances increase digestive secretions including enzymes and bile.The higher serum cholesterol content, which was observed in the PFA fed animals of the present study, may be the result of an increased lipid digestibility due to a higher secretion of bile and digestive enzymes.The improvement of FCR as well as the increase of blood nutrient concentrations supports this assumption, although it remains speculative as nutrient digestibilities have not been investigated in the present study.
Regarding the intestinal morphology, heavier chickens are generally associated with longer villi, greater villus width and higher villus surface area as compared to lighter ones (Adibmoradi et al., 2006;Incharoen et al., 2010).In the present study there were insignificantly increased villus heights and decreased crypt depths in the jejunum of birds receiving 150 mg/kg PFA compared to the control.Accordingly, the villus height: crypt depth ratio was significantly higher in the birds fed the PFA compared to the control.These findings were consistent to Adibmoradi et al. (2006) who reported that jejunal villus height was increased whereas crypt depths were decreased, leading to increased villus height: crypt depth ratio in birds fed graded levels of garlic meal.It has been suggested that longer villi would result in an increased surface area and higher absorption of available nutrients (Caspary, 1992;Yasar and Forbes, 1999).A higher absorptive capacity of the intestine of PFA fed animals is also supported by higher blood nutrient concentrations of those animals, as observed in the present study.However, although Jamroz et al. (2006) reported similar improvement of FCR due to the supplementation with a plant extract containing carvacrol, cinnamaldehyde and capsicum oleoresin, the authors could not find an effect of the plant extract on intestinal morphology in broilers at 42 d of age.Thus, for PFA containing essential oils as well as pungent substances further, eventually synergistic, effects or other modes of action cannot be ruled out.

Conclusion
In the present study, the addition of the phytogenic feed additive BIOSTRONG® 510 to broiler diets significantly improved feed conversion ratio.The observed improvement of feed conversion ratio may be caused by a combination of different effects, including enhancement of digestive secretions, antimicrobial effects of essential oils as well as enlargement of intestinal absorptive surface.However, further studies are necessary to evaluate the effects of various substances present in phytogenic feed additives and to clarify their specific modes of action.

Table 1 .
Ingredients and nutrient composition of experimental diets.

Table 2 .
Effect of the phytogenic feed additive on broiler performance during the starter and grower period (means ± SD).

Table 3 .
Effect of the phytogenic feed additive on some blood parameters in broilers (35 th d of age; means ± SD).

Table 4 .
Effect of the phytogenic feed additive on histomorphological parameters of the jejunum in broilers (means ± SD).Villus height and crypt depth in the jejunum of broilers fed diets with or without the phytogenic feed additive at 42 days of age, left control, right phytogenic feed additive.