Comparison of Different Herbal Additives on Immune Response and Growth Performance of Broiler Chickens

This experiment was conducted to compare the effects of four commercial herbal additives (Noviherb ® , Bioessence ® , Biostrong ® , and Novigrow ® ), and a commercial antibiotic (virginiamycin) on growth performance, carcass yield, visceral organs weight, thyroid hormones, and humoral immune responses of broiler chickens. Nine hundred day-old Ross 308 male broiler chicks were reared in litter-covered floor cages and distributed into five experimental groups with six replicates of 30 birds. Five dietary treatments tested were diets supplemented with Noviherb ® , Bioessence ® , Biostrong ® (each of them 100g/ton of diet), Novigrow ® (1000g/ton of diet), and virginiamycin (100g/ton of diet) as a positive control. Feed intake, body weight, mortality, feed conversion ratio, European broiler index, and feed cost per kilogram of body weight were measured during the experiment. Antibody titers against sheep red blood cells (SRBC), immunoglobulin G, immunoglobulin M, and plasma concentrations of thyroxine (T4) and triiodothyronine (T3) were evaluated at the age of 42 days. Then, four birds per replicate were killed to determine the relative weight of carcass and organs. The GLM procedures of SAS software and Duncan’s multiple range test were applied to analyze data in a completely randomized design with five treatments and six replicates of 30 birds per each. The effects of herbal additives on productive traits feed cost per kilogram of body weight and relative weight of organs were not significant in comparison with antibiotic treatment. Dietary inclusion of Noviherb ® , Biostrong ® , and Novigrow ® significantly decreased feed cost per kilogram of carcass compared with Bioessence ® or antibiotic treatments (p<0.05). The titers of IgG in Noviherb ® and virginiamycin were significantly higher (p<0.05) than in Biostrong ® and Novigrow ® fed groups. The plasma concentration of triiodothyronine was significantly decreased (p<0.05) by dietary inclusion of Noviherb ® and Novigrow ® compared to the antibiotic. In conclusion, all the evaluated herbal additives could act as a substitute for the growth-promoting antibiotic.


INTRODUCTION
Aside from the substantial role of phytogenic and medicinal compounds derived from plants in human nutrition for increasing appetite due to their functions as essence and odor, the herbal additives could also be applied in livestock feeding for animal well-being and growth improvement (Singh & Gaikwad, 2020). Considering the prohibition of antibiotics usage in poultry diets due to the ontogeny of microbial resistance, therefore, alternative substances for growth enhancers were necessary to be made and applied (Saleh et al., 2018). Meanwhile, different kinds of bioactive additives were used to optimize the gastrointestinal integrity of birds and to promote their performances and immune functions (Adedokun & Olojede, 2019).
Various phytogenic compounds showed pharmacological benefits and were broadly applied in human basil, lemon, tea, caraway, sage, and thyme) into broiler diet (100-500 g/ton) improved the weight gain, feed efficiency, and relative weight of breast and carcass (Khattak et al., 2014); meanwhile, blood biochemical parameters were not affected by the mentioned herbal mixture. Kini et al. (2016) reported that dietary inclusion of KiFAY (a commercial powder composed of garlic, onion, diatomaceous, and algal cell wall components) improved the performance of broilers with associated responses at thyroid hormones level (T 3 and T 4 ).
Numerous mixtures of medicinal plants which are commercially available for the poultry industry claim to have bio-functional advantages regarding growth performance and immunocompetence. The results of recent research have shown that plant essential oils can be produced and added to the diet using micro and nanoencapsulation technologies at low and economic levels in order to allow slow release upon ingestion and have a direct effect on the target tissues (e.g., intestine) (Hosseini & Meimandipour, 2018;Amiri et al., 2020;Lee et al., 2020). Despite examining the effects of some of these products individually, there is no enough data to compare the effects of these new (micro or nano encapsulated) forms with common forms of herbal additives. Therefore, the aim of the present study was to compare the effects of four commercial herbal additives, including nano encapsulated (Noviherb ® ) and microencapsulated (Biostrong ® ) forms and a commercial antibiotic on the growth performance, carcass yield, visceral organs, and immunity of broiler chickens.

Ethical Standards
The experimental methods used in the present research were ratified by the Animal Ethics Board of Animal Science Research Institute of Iran (Approval No., 08415).

Birds, Housing, and Rearing Conditions
The location of this experiment was the Research Poultry House of Animal Science Research Institute of Iran (Karaj, Iran). Nine hundred one-day-old broiler chickens (Ross 308, with equal gender proportion) were obtained from an industrial hatchery and randomly assigned to 30-floor cages after weighing. Three square meter space was considered for each cage (0.08 m 2 for each bird), and wood-shaving was used as litter to cover the floor of the cages. The initial temperature of the poultry house was kept at 33 ± 2°C and was regularly declined (2.4°C weekly) to get a constant temperature level with the range of 21-23°C at the age of 28 days. Through the period of the experiment, lighting regime and relative humidity were maintained in 23:1 h of light and dark cycle and 50%-60%, respectively.

Acquisition and Compounds of the Additives
Noviherb® was obtained from Soroush Sabz Alborz Company (Karaj, Iran). It is an herb-base addi-

Diet Formulation and Experimental Design
Before formulating the experimental diets, the chemical composition of main feed ingredients, including soybean meal and corn, were analyzed following the AOAC procedures (AOAC, 2012), and resulting data were applied to formulate the experimental diets (Tables 1 and 2). Nine hundred Ross 308 male broiler chicks on the first day of age were reared in cages with litter-covered floor and were distributed into five experimental groups with six replicates of 30 birds each. Five treatments were diets supplemented with Noviherb® (100g/ ton of diet), Bioessence® (100g/ton of diet), Biostrong® (100g/ton of diet), Novigrow® (1000g/ton of diet), and virginiamycin (100g/ton of diet) as a positive control. A three-phase feeding program was applied, namely, starter (days 1 to 10), grower (days 11 to 24), and finisher (days 25 to 42) with basal diets were formulated for the respective period. All diets in each phase contained similar amounts of energy and protein, according to Ross 308 recommendations (Aviagen, 2014). The birds were given feed ad libitum and free access to drinking water during the experiment.

Data and Sample Collection
Parameters regarding performance like feed intake (FI) and body weight (BW) were recorded at ages of 10, 24, and 42 days. Mortality was recorded daily in order to calculate live ability. Also, feed conversion ratio (FCR) and FI were calculated during days 1 to 10, 1 to 21, and 1 to 42 of the rearing periods. On day 42, 24 birds from each treatment (four birds per pen which had close body weight to the average of the pen) were chosen. The birds were weighed and killed after four hours of fasting; then, they were dissected, and the visceral organs were moved to measure the relative weight of carcass and organs. Weighing scale (0.001 g, model DJ-V 320A; AND Weighing, China) was used to measure the weight of carcass, legs, breast, and organ weights. At the end of the experiment, the European broiler index (EBI) was calculated based on the formula of Marcu et al. ( 2013) as follows: EBI= [livability (%) x average daily gain (g)] / [feed conversion ratio (g/g) x 10].
Total feed cost was measured by multiplying total feed consumed by the price per kilogram of each dietary treatment. Feed cost per kilogram of body weight was calculated as total feed cost divided by live body weight at the end of the experiment (day 42). Feed cost per kilogram of the carcass was calculated as total feed cost divided by carcass weight at the end of the experiment.
At the ages of 28 and 35 days, one mL of 5% suspension of sheep red blood cells (SRBC) was intravenously administrated to four birds per replicate. Blood samples were collected one week after the second administration (Peterson et al., 1999). Blood samples were centrifuged at 3000×g for 15 minutes, and the prepared sera were stored at -20°C for subsequent analyses. The inactivation of each serum sample was performed at 56°C for 30 min, and then sera were analyzed for total titer of anti-SRBC antibodies according to the method of Moslehi et al. (2019). Briefly, each inactivated serum was titrated in order to evaluate the total and mercaptoethanol (ME)-resistant (IgG) anti-SRBC antibody titers. Titers of ME-sensitive (IgM) antibodies were gained by subtracting IgG antibodies level (titer) from total antibodies. All data for antibody titer were reported in terms of log 2 .
At 42 days of age, blood samples of four birds in each replication were taken via wing vein. Blood samples were subsequently centrifuged (1,000 × g for 15 min) to obtain plasma and retained at −20°C for further analysis (Kamely et al., 2015). Plasma concentrations of thyroxine (T 4 ) and triiodothyronine (T 3 ) were measured by the commercial laboratory kit (Pishtaz Teb, Tehran, Iran) through the enzyme-linked immunosorbent assay.

Statistical Analysis
The GLM procedures of SAS 9.1 software (SAS, 2003) were applied to analyze data in a completely randomized design. Percentage data with high enough CV value were transformed to arcsine before analysis. The pen of birds functioned as the experimental unit. When treatment indicated significant effect, it was continued to Duncan's multiple range test for the comparison of the means at 5% probability.

Growth Performance
The results of herbal additive effects on the performance of broiler chickens are summarized in Table 2. As shown in this table, the effects of four herbal additives on BW, FI, FCR, liveability, EBI, and feed cost per kilogram of BW were not different among all treatments. At the age of 42 days, FCR in birds that received herbal additive treatments was numerically lower, but it was not statistically different than those given antibiotics.

Carcass Characteristics
The effects of herbal additives on carcass yield and internal organ weights of broiler chickens in comparison to antibiotic treatment are presented in Table 3. The carcass yield and relative weights of breast, leg, heart, liver, gizzard, and intestine were not affected by dietary treatments. However, dietary inclusion of Noviherb®, Biostrong®, and Novigrow® were significantly decreased (p<0.05) feed cost per kilogram of carcass compared with Bioessence® or antibiotic treatments.

Humoral Immunity
The effects of dietary treatments on humoral immune responses of broiler chicks on day 42 are indicated in Table 4. Regarding SRBC and IgM, no significant difference was observed among dietary treatments; however, the titer of IgG was affected by the inclusion of additives (p<0.05). The titers of IgG in groups fed with Noviherb ® , virginiamycin, and Bioessence ® were significantly higher (p<0.05) than those receiving Biostrong ® .

Thyroid Hormones
The results of the herbal additive effects on thyroid hormone concentrations of 42 days old broiler chickens in comparison to the antibiotic are displayed in Table 5. As shown in this table, the concentration of T 3 and the ratio of T 3 to T 4 were significantly (p<0.05) influenced by  In addition, the ratio of T 3 to T 4 was significantly lower in all herbal additive groups than antibiotic-treated birds (p<0.05).

DISCUSSION
The results of this research showed that the effects of four evaluated herbal additives on productive traits (BW, FI, FCR, liveability, EBI, and feed cost per kilogram of BW) were not significantly different in comparison with antibiotic treatment, suggesting that the use of antibiotics as a growth promoter can be replaced by any of these additives.
The dietary inclusion of antibiotics is an old used strategy in order to promote the performance of broiler chicks (Hussein et al., 2020). Subtherapeutic, in-feed antibiotics could improve body weight gain up to 8% and decrease the feed conversion ratio (feed intake/body weight gain) up to 5%, both compared with an antibiotic-free diet during 1-42 days of age (Gadde et al., 2018). These researchers also reported that dietary supplementation of virginiamycin, increased chicken body weight gain by 10.1% between days 1 and 21 of age compared with control groups. Furthermore, a noticeable correlation has been reported between the inclusion level of avilamycin in the diet and the reduction in the mortality rate (Paradis et al., 2016;Mwangi et al., 2018). Since the forbidding of such antibiotic growth promoters, alternative approaches have been offered, such as the dietary addition of organic acids, prebiotics, probiotics, and Phyto biotics (Mora et al., 2020).
Phyto biotics have been reported to possess capabilities such as the improvement of health and performance (Diaz-Sanchez et al., 2015;Clavijo-López & Vives-Florez, 2018). Plant-based essential oils (individually or combined together) have been applied as food and feed additives to boost the immune responses of humans and animals against different pathogens (Bajpai et al., 2012;Ali, 2014;Hassan et al., 2016;Hussein et al., 2020).
Carvacrol and thymol, the two basically similar main components of oregano and thyme, as well as an essential oil, were found to have a synergistic effect on the physiological state of broilers (Hashemipour et al., 2013). Moreover, p-cymene found in ajwain and savory, a biological precursor of carvacrol, was found to have a higher favorite for liposomal membranes, thereby producing more expansion. By this mechanism, cymene maybe enables carvacrol to be more easily transported into the cell so that a synergistic effect is attained when the two are used together (Zeng et al., 2015).
Bioessence ® contains six medicinal plants essences including thyme, oregano, anise, fennel, savory, and dracocephalum. Some of the major chemical compositions found in anise seed include p-anisaldehyde, estragole, anise alcohol, limonene, anethole, and Note: a-d Means in the same row with different superscripts differ significantly (p<0.05). SEM= Pooled standard error of the mean. ¹= The data for antibody titer was reported in terms of log2; ²= Each herbal additive was supplemented at the level of 100g/ton of diet, except Novigrow® which was supplemented at the level of 1000g/ ton; ³= Virginiamycin was included as a positive control and supplemented at the level of 100 g/ton diet. pinene, but anethole is a main component (Sun et al., 2019). Estragole and Trans-anethole were found to be the main components of fennel seeds essential oils (Diao et al., 2014). According to the results of Miladi et al. (2013), the essential oils of savory were dominated by γ-terpinene, mono-terpenic hydrocarbons p-cymene, and carvacrol. Farimani et al. (2017) noted that the major compound in the essential oils of dracocephalum is limonene. These researchers also showed antibacterial properties of this plant. The antimicrobial activity of savory is originated from carvacrol, thymol, and other monoterpenoid hydrocarbons (Miladi et al., 2013). A number of studies indicated the synergistic antibacterial effects of essential oils containing anethole, estragole, limonene, and carvacrol in combination together (Bassole & Juliani, 2012;Auezova et al., 2020). So, we can conclude that Bioessence ® is established upon compounds having an antimicrobial activity that can act as an alternative for antibiotics. Biostrong ® 510 is comprised of a microencapsulated form of herbal essential oils (mainly thyme, star anise, and quillaja bark). Compounds like α-and β-pinene, α-and γ-terpineol, limonene, linalool, estragole, and trans-anethole present in the essential oils of star anise are shown to possess various antibacterial activities (Aly et al., 2016;Luis et al., 2019). Supplementing Biostrong in diet improved growth rate, meat production, and feed efficiency of Cobb 508 broiler chickens (Lavrentyev et al., 2019). Therefore, this product can also be used as an alternative for the growth-promoting antibiotic.
Combinations of certain essential oils (estragole, limonene, anethole, thymol, and carvacrol) exhibited a synergic effect against foodborne pathogens and also a vital reduction in their individual minimum inhibitory concentrations (MIC) against foodborne pathogens. Thyme essential oils presented the lowest singular MIC, but its usage in the mixture declined the MIC of the other essential oils (Garcia-Diez et al., 2017). The capability of Bioessence ® and Biostrong® 510 for the substitution of antibiotics could be related to synergism between their individual bioactive components and the aforementioned mechanism.
Novigrow ® is made based on lactose, yeast cell wall, lactic acid, formic acid, and some herbal compounds like turmeric, garlic, and cinnamon powder. Vicente et al. (2007) applied lactose as a prebiotic and showed that dietary addition of products containing lactose and Lactobacillus spp. had positive effects on feed conversion ratio and body weight gain of turkeys infected with Salmonella. Yalcin et al. (2014) reported augmented humoral immune response, improved growth performance, and reduced abdominal fat by dietary inclusion of yeast cell wall. Based on the results of Fascina et al. (2012), organic acids have growth-promoting properties and can be used as substitutes of antibiotics. Navidshad et al. (2018) indicated that chemical components of garlic (including allicin) have beneficial effects for animals, including growth-enhancing and antimicrobial effects. El-Hack et al. (2020) reviewed the antimicrobial properties of cinnamon and concluded that it could be applied as a replacement for antibiotics for more well-being, financial, and environmental aspects of poultry production. In conclusion, we can express that Novigrow ® also possesses antibiotic-like properties.
Carcass yield and relative percentage of breast, leg, heart, liver, gizzard, and intestine were not influenced by dietary treatments. It has been hypothesized that dietary antibiotic supplementation reduces the relative weight of the intestine (Gunal et al., 2006). In the current study, there was no major difference between herbal additives and antibiotics regarding carcass yield and organ relative weight, which may imply antibiotic-like properties of applied herbal additives. Additionally, the lower feed cost per kilogram of the carcass in birds that received Noviherb®, Biostrong®, and Novigrow® in comparison to the antibiotic may imply the financial benefit of using herbal additives for broiler producers (El-Hack et al., 2020).
In the present study, the IgG titers in Noviherb ®received group were similar to that given virginiamycin and were higher than birds receiving Novigrow ® or Biostrong ® . Yakhkeshi et al. (2011) reported that application of Primalac® and virginiamycin resulted in the maximum and minimum antibody titers against SRBC, respectively. Increased immune responses have been noted by using herbal extracts in diets (Mathivanan & Kalaiasari, 2007) and are in agreement with the data obtained in this study. According to Cook & Samman (1996), herbal extracts augment immune response by promoting vitamin C action. For instance, Sangrovit® is a herbal additive known to possess immunomodulatory properties (Karimi et al., 2014). It has been noted that Sangrovit® motivates the activity of phagocyte and therefore endorses defensive responses against diseases in broilers (El-Sheikh et al., 2018).
In our study, the concentration of T 3 was reduced in birds with dietary inclusion of Noviherb ® and Novigrow ® compared to the antibiotic. Also, the T 3 to T 4 ratio was lowered in all the birds fed with herbal additive than the antibiotic-receiving group. In the study of Sadeghi & Moghaddam (2018), the concentration of thyroid hormones in broiler chicks that received medicinal plants had a tendency to increase at the age of 42 days. This result was in contrast with the present study. On the other hand, the reducing effect of some flavonoids on thyroid function is reported in rats by Chandra & De (2010). These researchers indicated that catechin prevents the activity of thyroperoxidase and decreases the serum levels of T 3 and T 4 .

CONCLUSION
In conclusion, dietary inclusion of the evaluated herbal additives can be considered as substitution alternatives to growth-promoting antibiotic. The IgG titers in birds receiving Noviherb ® indicate the same value as that given virginiamycin and are higher than that fed with Biostrong ® or Novigrow ® . Feed cost per kilogram of carcass is decreased in birds consumed Noviherb®, Biostrong®, and Novigrow® in comparison to that received growth promoter antibiotic.