Growth, Carcass Composition, Haematology and Immunity of Broilers Supplemented with Sumac Berries (Rhus coriaria L.) and Thyme (Thymus vulgaris)

Simple Summary Widespread use of antibiotics is known to cause resistance in bacteria, yet they are routinely used to improve growth performance in meat chickens. We investigated two medicinal plants, thyme and sumac berries, for their ability to function as an alternative to antibiotics in the diet of broilers. The actual plants or parts of the plants have rarely been fed before as supplements, mostly their extracted oils have been used instead. In our study, they were fed at 1–3% of the diet to investigate if the effects were dependent on the dose. We found evidence of improved immune systems with both medicinal plants when the chickens were tested for resistance to Newcastle Disease and influenza. Sumac berries, and to a lesser extent thyme, reduced the fat content of the birds’ abdomen, by 62 and 41%, respectively, reflecting an observed reduction in lipids in the blood. It is concluded that these two medicinal plants offer potential to replace antibiotics in the diet of meat chickens, as well as offering benefits in reducing the fat content of the birds. Abstract Alternatives to antibiotics as growth promoters for broilers could reduce bacterial resistance to antibiotics, while at the same time maintaining growth and improving carcass composition. We investigated the benefits of adding the medicinal plants sumac and thyme at 1, 2 or 3% of the diet for male Ross broiler chicks, with four replicates of ten birds in each treatment group and a Control. Feed intake was reduced for chickens fed the sumac supplements, and, at the two higher doses, defeathered body weight was also reduced. Abdominal fat was reduced by 41% in chickens fed thyme and 62% in those fed sumac. This reflected reduced low density lipoproteins in their blood, and in higher dose thyme treatments and all sumac treatments, reduced high density lipoproteins in blood. Apart from this, there was little effect of the supplements on carcass composition. Blood glucose was reduced in the supplemented chickens. There was evidence of higher antibody titers to Newcastle disease and influenza in supplemented chickens. It is concluded that both thyme and sumac offer potential to reduce fat content and improve disease responsiveness in broiler production systems.


Introduction
In the broiler industry, birds are often reared in confinement at high stocking densities, which can predispose them to rapid disease transmission. Potentially damaging feed additives such as antibiotics, growth promoters, and anti-coccidial drugs are routinely used to enhance growth rates, combat diseases, with similar body weights (mean 44.0 ± 1.3 g) were randomly assigned to four replicates of seven treatment groups of 10 birds/group from 1-42 days of age. Growth parameters were measured on all birds. From these seven treatment groups, 1 bird/replicate was used for blood sampling, for immunity parameters a different bird was used from each replicate, and for carcass samples yet another bird was used from each replicate. The birds in the control group were given the basal diet, while those in treatment groups were given supplements as detailed below: Treatment 1: Basal diet (control); T1: Basal diet + thyme powder at 1% from days 29-42 T2: Basal diet + thyme powder at 2% from days 29-42 T3: Basal diet + thyme powder at 3% from days 29-42 S1: Basal diet + sumac powder at 1% from days 29-42 S2: Basal diet + sumac powder at 2% from days 29-42 S3: Basal diet + sumac powder at 3% from days [29][30][31][32][33][34][35][36][37][38][39][40][41][42] The basal diet consisted of a three-phase feeding program: starter feed from d 1-7, grower feed from d 8-24, and finisher feed from d [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42]. The ingredients and nutrient composition of the diets are shown in Table 1. Feed and water were supplied ad libitum throughout the experimental period. The diets met or exceeded Ross 308 catalogue recommendations [27,28]. Sumac fruit and thyme plants were purchased as a single batch at Rasht market, Iran, dried and ground into a powder before adding to the diet. The experiment was repeated four times and the total number of birds used in the study was 280 (70 × 4). The broiler groups of ten were kept in cages (1.0 × 1.0 × 0.5 m), each fitted with an individual feeder and nipple drinker. The cages were kept in a windowless, environmentally-controlled room, with room temperature initially maintained at 32 • C, declining to 22 • C, as appropriate for the age of the bird, and a light cycle of 23 hour light, 1 hour dark/day. Treatment groups were allocated to cages so that they were equally distributed in the room to minimize any cage position effect. Chickens were subjected a routine vaccination program. Briefly, the birds were vaccinated against infectious Bronchitis virus (IBV) (H120; 1st day of age), Newcastle disease virus (NDV) (8th and 21st day of age), influenza (1st day of age), and Gumboro disease (IBD071IR; 14th and 23rd day of age).

Growth Performance
On days 28, 35, and 42 body weight and feed intake were measured. On the same days, feed conversion ratio, average daily energy intake, gain to metabolizable energy ratio, average daily protein intake, and gain to protein intake ratio were calculated and corrected for mortality.

Blood Serum Parameters
At the end of the growth period (day 42), blood samples were collected from four birds per treatment (1 bird/replicate group) from the wing vein. These blood samples of 1 ml/bird were collected into EDTA tubes and then centrifuged (3,000 rpm × 10 min) at room temperature. Then, plasma was stored at −20 • C until analysis. Biochemical analyses (glucose, uric acid, total cholesterol, triglycerides, cholesterol linked to high density lipoproteins (HDL), cholesterol linked to low density lipoproteins (LDL), and HDL/LDL) were performed using standard protocols of commercial laboratory kits (Pars Azmoon Co., Tehran, Iran) [29].

Immune Response
Four birds per dietary treatment (1 bird/replicate group) were chosen at random for blood sample collection from the brachial vein. Blood serum was separated by centrifugation (3000 rpm × 15 min) and antibody titers against Newcastle Disease Virus (NDV) and avian influenza (AI) measured, respectively, at 14 and 28 d after the related vaccine injection, by the hemagglutination-inhibition test [30]. For this test, in U-bottom microtiter plates, two-fold serial dilutions of heat-inactivated (at 56 • C) serum were made with phosphate-buffered saline (PBS) (0.01 mol/L; pH 7.4) for total antibody. All antibody titers were recorded as log 2 of the highest dilution of serum that agglutinated an equal volume of a 0.5% suspension in PBS [31].

Carcass Measurements
At the end of the study (day 42), four birds per dietary treatment (1 bird/replicate group) were selected that were approximately the average body weight of the group. They were killed by cervical dislocation to evaluate carcass traits. The defeathered carcasses were weighed before and after removal of the head and drumsticks. Viscera and abdominal fat were then removed and the carcass yields (with the abdomen empty), and the relative weights (expressed as a percentage of the eviscerated carcass) of abdominal fat, anatomical parts (breast, drumsticks, wings, head, neck, and notarium), internal organs (heart, kidneys, and pancreas) were calculated. The relative weights of organs of importance to the immune system (thymus, liver, spleen, and bursa of Fabricius) were also determined.

Statistical Analysis
Cages were the experimental unit for performance traits, while the individual bird was the experimental unit for carcass and organ characteristics, and immunity and hematological traits. The model assumptions of normality and homogeneity of variance were tested using Shapiro Wilk and Levene tests, respectively. Data were analyzed by the ANOVA option of the general linear model of SAS/STAT software (SAS Institute Inc., Cary, NC, USA) for a completely randomized design with dietary additive as the main effects. The statistical model used was: where: Y ijk = response variables from each individual replication or pen, µ = the overall mean, T i = the effect of dietary additive, R ij = the inter-experimental unit (replications) error term, E ijk = the intra-experimental unit error term. Means were compared for significant differences using the LSMEANS option of SAS version 8 (SAS Institute Inc., Cary, NC, USA). Statistical significance was established at p ≤ 0.05.

Feed Intake, Growth, and Body Composition
Feed intakes were reduced in T2 and S1, S2 and S3 treatments compared with the Control treatment ( Table 2). Weight gain, feed conversion ratio (Table 2), and live body weight (Table 3) were not affected by treatment. Defeathered body weight was reduced for the two higher sumac feeding levels (S2 and S3) ( Table 3). Carcass weight was reduced for S2 compared with S1. The abdominal fat was reduced in the thyme treatments and even more reduced in the sumac treatments, with no differences due to level of supplement feeding (Table 4). Heart, neck (Table 4), and testes (Table 5) weights were not affected by treatment, nor were the weights of economically valuable parts-breast, drumstick, and wings-or the less valuable parts-head and lungs (Appendix A Tables A1 and A2). Relative kidney weight increased in S3 compared with S1 and the Control treatments (Table 5).

Haematology and Response to Vaccination
Blood glucose concentration was higher in the Control treatment, compared with the 2 and 3% thyme treatments and the 1 and 3% sumac treatments. Total cholesterol was reduced in the sumac treatments compared with the Control (Table 6). Compared with the Control, T1 and T2, the treatments T3 and S1, S2, and S3 all had reduced triglycerides. Similarly, compared with the Control, the treatments T2, 3 and S1, S2 and S3 all had reduced high density lipoproteins and all treatments had reduced low-density lipoproteins.
Compared with the Control, all T treatments and S2 had higher Newcastle disease antibody titers, and T3 and S3 had higher influenza antibody titers (Table 7).

Feed Intake and Growth
Feed intake was reduced by including sumac in the diet at any level, yet only the two higher sumac feeding rates reduced body weight. Previous studies have found reduced body weight at higher feeding rates than in this study [23]. Other studies with poultry have found reduced egg weight at relatively low levels [26], confirming reduced production in chickens. Thyme also reduced feed intake in our study, but only at 2%. Conversely Zhu et al. [32] found no effect of thyme oil on the feed intake of Luhua chickens, but the growth rate of the chickens was increased when adding thyme oil. Kalantar et al. [33] reported improvements in feed efficiency with thyme, including benefits in dressing weight. Zhu et al. failed to find any effect on dressing % [32].

Body Composition and Haematology
The abdominal fat weight was affected primarily by the supplements, regardless of dose, being reduced by 41% for chickens fed the thyme supplements and 62% for chickens fed the sumac supplements. This reflects reduced low density lipoproteins in the blood in all supplemented chickens, compared with the control group. Aghazadeh et al. have found reductions in total and LDL cholesterol in chickens supplemented with thyme extract [34]. We also found reduced HDL in treatments T2 and T3, in contrast to Aghazadeh et al. who did not find any effect of thyme extract on HDL. The reduction in circulating triglycerides in T3 and S1, 2 and 3 is almost certainly connected with reduced abdominal fat deposits and was also found by Aghazadeh et al. [34]. Circulating plasma lipids are responsible for nearly all of the fatty acids in adipose tissue and de novo synthesis of fatty acids is limited. The latter is enhanced by insulin and reduced by thyroxine and glucagon. The jejunum is the main site of lipid absorption in chickens. It is likely that the reduction in abdominal fat and circulating triglycerides was due to the inhibition of hepatic 3-hydroxy-3-methyle glutaryl coenzyme A (HMG-CoA) reductase activity that thyme produces. This is a key regulatory enzyme in cholesterol synthesis. Thyme also has saponins, which complex with cholesterol and prevents its absorption. In our study thyme also reduced blood glucose, whereas in other research it has increased it, which was believed to occur for the support of gluconeogenesis [35].

Responses to Vaccination
Compared with the Control, the response to vaccination appeared dependent on concentration, with, at least for response to the avian influenza vaccination, improved titres at the higher concentrations of inclusion of thyme and sumac. Other researchers have noted that thyme, either alone or in combination with other herbs, produced no change in response to vaccination for Newcastle Disease, but low levels of suplementation were used [11]. A multiple herbal supplement containing thyme, oregano, chamomile, and peppermint essential oils has improved vaccine titers against avian influenza and Newcastle disease [36], and another supplement with thyme, cinnamon, and turmeric has shown similar responses in titer in response to Newcastle Disease vaccination [6]. However, in such studies it is not possible to determine which herbs were causing the response. According to the review of Papatsiros et al. [37], thymol and oregano both have medium antimicrobial activity, but cinnamon has strong activity.

Limitations of the Study
It is uncertain whether we used the supplements for the right length of time. Most previous work used thyme or sumac from day 1-42 of age, but there is significant cost to their inclusion for such a long period of time. Therefore, we reduced the feeding period to days 29-42 of age, in the expectation that we could get the same effect on carcass quality but at reduced cost. This appears to have been the case from our results. Further research should investigate the minimum time period to get the maximum benefit.
A second limitation is that we measured only titers in response to vaccinations, not the diseases themselves. Testing our results to examine whether disease prevalence might be reduced in farms not normally vaccinating would be useful, but this could only be done over a long period, during which time there might be an outbreak.

Conclusions
Both thyme and sumac berries had benefits in reducing fat deposition in growing broiler chickens, particularly the latter. Although much less is known about the benefits of sumac than thyme, for many hematological parameters, for example triglyceride and total cholesterol concentrations in the blood, it appeared to be more potent. However, thyme appeared to be more potent than sumac against Newcastle disease, although both had similar activity against influenza.   Table A1. Mean (±SEM) of economically valuable parts in relation to eviscerated carcass at 42nd days of age in Ross 308 broilers fed diets containing 0, 1, 2 and 3% Thyme (T3) and sumac from days 29-42.