Effects of Ground Thyme and Probiotic Supplements in Diets on Broiler Performance, Blood Biochemistry and Immunological Response to Sheep Red Blood Cells

Abstract

A trial was conducted to study the effects of the aromatic plant thyme, a commercial probiotic (Protexin) and avilamycin on broiler performance, blood biochemical parameters and also the antibody response to sheep red blood cells. A total of 750 broilers were assigned into five replicate groups for each of five dietary treatments, namely; control (C), 2.5 mg/kg avilamycin (AB), 0.1 g/kg commercial probiotic (P), 5 g/kg ground thyme (T1), and 7.5 g/kg ground thyme (T2). In general, body weight, feed consumption and feed conversion ratio were not affected by dietary treatments compared to the control birds (P>0.05). Birds fed the P supplemented treatment had the greatest serum protein levels (P<0.001) and highest albumin levels (P<0.001) when compared with control birds, while the birds fed T2 had the lowest (P<0.001). Dietary supplementation reduced (P<0.001) cholesterol and triglyceride concentration in serum of broilers, with the effect were more noticeable by P supplements. Moreover, blood calcium and phosphorus concentrations were higher (P<0.001) in birds fed the P-supplemented diet compared to the birds fed the control diets.

This study suggests that probiotic supplementation in particular, and to an intermediate extent ground thyme supplementation in diets of broiler, resulted in chicks with favorably improved blood biochemical parameters and mineral utilization, compared to the birds fed diets supplemented with avilamycin or without any supplementation.

Keywords:

• Broiler
• Blood parameters
• Dietary thyme
• Probiotic
• SRBC antibody response

Introduction

For many years, several antibiotic growth promoters (AGP) have been effectively used as additives in poultry production. However, the overuse of these compounds has led to the emergence of antibiotic-resistant bacteria. Pressures are increasing from consumers, in their requirement for safe, healthy and nutritious foods which are produced naturally. From January 2006, all sub-therapeutic AGP have been banned in the European Union (European Commission, 1998). Removal of AGP from diets requires the industry to look for various alternatives to maintain or improve the health and performance of broilers. However, most of the alternatives can exert growth-promoting effects and some of the effects are comparable to those of AGP, but these growth-promoting effects are very variable; under certain circumstances the alternatives can even negatively affect the performance (Yang et al., 2009).

Plant-based additives are perceived to be safer and healthier than synthetic AGP. Herbs and phytobiotics are incorporated into poultry diets to stimulate or promote a more effective use of feed nutrients, which may subsequently result in a more rapid gain in body weight and improved feed conversion efficiency. Moreover, active components of herbs may improve digestion and stimulate broiler immune function (Ghazalah and Ali, 2008). Thyme (Thymus vulgaris) is known in Iran as shirazian thyme, and is a popular medicinal plant in the region. The active components of thyme are thymol and carvacrol, which are known for their antioxidant and antibacterial properties (Ouwehand et al., 2010). On the other hand, the birds received diet supplemented with probiotics showed the enhanced systemic antibody response to some antigens (Haghighi et al., 2005). The in vivo reports on the effect of thyme on blood parameters and immunity are very limited, whereas the benefits of probiotics on the immune system are becoming more widely accepted. The purpose of current study was to investigate the effect of substitution of ground thyme as a phytobiotic and a probiotic in broiler diets versus avilamycin on growth performance, blood biochemical parameters, and the antibody response to sheep red blood cell.

Materials and methods

Dietary treatments

A total of 750 one-day-old male Cobb broiler chicks were obtained from a local hatchery and used in the experiment. The chicks were randomly allocated in groups by weight to one of five replicates for a total of five dietary treatments in a completely randomized experimental design. Each replicate consisted of 30 chicks housed in a floor pen (3×1 m). The basal diet consisted of a maize-soybean meal control ration (C), was supplemented with; 2.5 mg/kg of avilamycin (AB); 0.1 g/kg of a commercial probiotic (Protexin, consisting Lactobacillus acidophilus, L. bulgaricus, L. plantarum, L. rhamnosus, Bifidobacterium bifidum, Candida pintolopesii, Enterococcus faecium, Aspergillus oryzae and Streptococcus thermophilus with minimum 2×10⁹ CFU g¹ powder, supplied by Nikotech Inc., Tehran, Iran); 5 g/kg ground thyme (T1; Thymus vulgaris supplied by Animal Science Research Institute, Karaj, Iran); 7.5 g/kg ground thyme (T2), to form the four test diets. The Protexin was stored at 4°C and incorporated into the feed each day to ensure viable bacterial cells in the feed throughout the experimental period. The viability of the bacterial cells was checked every two week to ensure that the concentration of these viable bacterial cells remained at 10⁸ CFU/kg of feed. The test diets were prepared in mash form and formulated to be adequate in all nutrients, according to the National Research Council (1994) (Table 1). Food and water were provided ad libitum and the chicks were provided with artificial fluorescent illumination for 23 hours/day. The experimental diets were offered from day 1 to the end of the experiment at 42 days of age.

Traits

Body weight and feed consumption

All chicks were weighed individually, and their feed consumption recorded weekly during the experiment.

Serum biochemical studies

At 21 days of age, 5 mL of blood was collected from the brachial vein of 2 birds from each pen. The concentrations of total protein, calcium, phosphorus, albumin, globulin, triglyceride and cholesterol in serum samples were subsequently analyzed using colorimetric methods on an automatic biochemical analyzer (RA-1000, Bayer Corp., Tarrytown, NY), following manufacturer instructions for the corresponding reagent kit (Zhongsheng Biochemical Co., Ltd., Beijing, China).

SRBC response

At 28 days of age, 3 birds from each pen were inoculated with a volume of 0.2 mL from a 7% suspension of sheep red blood cell (SRBC). At 7 days post-inoculation (35 days of age); SRBC-inoculated birds were bled through the brachial vein, then the antibody titers (log 2) were measured by the microtiter haemagglutination procedure (Wegmann and Smithies, 1966). The reciprocal of the highest dilution with complete agglutination was taken to represent titer.

Statistical analysis

Data was analysed using SPSS version 11.0 (SPSS GmbH, München, Germany). The Kolmogorov Smirnov test was used to test the normal distribution of the data. One-way ANOVA and Duncan’s multiple range test were used to compare treatments for each parameter. Statements of statistical significance were based on P≤0.05.

Results and discussion

Previous studies reported varying effects of herbal plants or their extracts, probiotics and organic acids on broiler performance (Yakhkeshi et al., 2011). In the present study, feed intake tended (P=0.086) to be reduced by increasing the concentration of thyme in diets for broilers at 14 days (Table 2). According to the literature, the effects of plant extract on feed intake are inconsistent (Cross et al., 2007; Bolukbasi et al., 2006; Calislar et al., 2009; Kirkpinar et al., 2011). It has been shown that when relatively high amounts (5 g/kg) of thyme essential oil were added into diet, early feed consumption (8-14 days) was decreased in broilers, but adaptation by the birds lessened the negative effects of thyme on feed intake by birds as they got older (Cross et al., 2003). Similarly, results in the present study confirmed the negative influence of high concentrations of ground thyme herb (7.5 g/kg) on feed intake at early stages in the life of the chick (Table 2). It can be hypothesized that this negative effect may be due to low diet palatability when thyme is included.

Body weight was not affected by any of the dietary treatments over the course of the study (Table 2). Several factors such as genotype, housing, hygienic conditions, management, feeding system and diet affect BW gain. Previous studies have reported the beneficial effects of antibiotic, probiotic (Meimandipour et al., 2010) and phytogenics (Çiftçi et al., 2005; Simsek et al., 2007) on broiler performance. As alternatives to AGP, natural feed additives can help to promote better intestinal health, increase the production of digestive enzymes, and can improve dietary digestion and absorption to significantly impact chick performance, particularly when subjected to stressful environmental condition (disease, high temperature, poor feeding and management) (Meimandipour et al., 2010; Lee et al., 2003b; Zulkifli et al., 2000; Mountzouris et al., 2007). In the present study, good quality chicks were selected and reared in a controlled environment system, resulting in final chick mortality rates below 5%. There was no effect of dietary treatment on chick mortality. As shown in Table 2, birds fed antibiotic tended (P=0.087) to lower FCR compared to control group at 42 days of age.

Serum protein is the total amount of protein in the blood in which albumin is the most abundant. Albumin helps keep the blood from leaking out of blood vessels as well as to carry some medicines and other substances through the blood that is important for tissue growth and healing. Low total protein levels can suggest a liver disorder, a kidney disorder, or a disorder in which protein is not digested or absorbed properly. In the present study, dietary probiotic and thyme supplementation adversely influenced serum protein and albumin concentrations, with highest serum protein and albumin concentrations in the P-fed group compared to those birds fed the AB and C treatments (Table 3). Several authors have identified that probiotic supplementation in diets increased serum protein and albumin concentrations, in broilers (Agawane and Lonkar, 2004, Capcarova et al., 2011), piglets (Harding et al., 2008) and fish (Zhou et al., 2010). Harding et al., (2008) reported that probiotics did not stimulate gastrointestinal protein synthesis or reduce the severity of intestinal colitis, but reported an unidentified signaling mechanism between the gut and liver which could be responsible for the probiotic-induced increase in liver protein and plasma protein synthesis. Furthermore, dietary probiotic supplementation may increase the serum protein and albumin concentrations by increasing iron absorption in the lower intestine (Eizaguirre et al., 2002). Alternatively, the lowest decrease in serum protein and albumin, which was dependant on the level of dietary supplementation, was observed in the broilers fed different levels of dietary thyme supplements, in the present experiment. This finding supports the results of previous research which investigated the bioavailability of iron from local plants in Sprague-Dawley rats, where the researchers concluded that thyme supplementation with high iron content was absorbed most efficiently but utilized least efficiently in the rats, resulting in a decreased serum hemoglobin concentration (Abu-Jadayil et al., 1999).

In this study, birds fed the dietary probiotic supplements had lowest cholesterol and triglyceride concentrations (P<0.05), which was followed by T2, T1 and antibiotic groups (Table 3). Nutritional therapies such as probiotics have been suggested to be helpful in the management of elevated cholesterol. Some probiotic strains such as Lactobacillus acidophilus can possess the ability to hydrolyze bile salts (BSH), enabling the cholesterol levels to be reduced. Nguyen et al. (2007) fed 10⁷ CFU/g Lactobacillus plantarum on a daily basis to hypercholesterolemic mice, demonstrating that probiotic treatment lowered the serum cholesterol and triglycerides by 7% and 10% compared to the controls, respectively, potentially due to a direct breakdown of cholesterol and the deconjugation of bile salts. Thyme supplementation has also reduced blood cholesterol and triglyceride concentrations (Table 3). Abdolkarimi et al. (2011) added thyme extract in the drinking water of broilers, and reported that this significantly decreased plasma low density lipoprotein (LDL) cholesterol, total cholesterol and also triglyceride. These authors reported the effect in animals fed diet or water containing thyme could have been due to a suppressing effect of thymol or carvacrol on hepatic 3-hydroxy-3- methyl glutaryl coenzyme A (HMG-CoA) reductase, the ratelimiting enzyme of cholesterol biosynthesis and/or responsible for the formation of insoluble saponin-cholesterol complexes in the gastrointestinal tract of the broiler (Case et al., 1995; Lee et al., 2003a). Thyme contains saponins, which form insoluble complexes with cholesterol in the digesta and also inhibits the intestinal absorption of endogenous and exogenous cholesterol (Oakenfull et al., 1990).

The birds fed diets containing probiotic had the highest concentrations of blood calcium and phosphorus, while those receiving the control diet were lowest and the birds fed thyme supplements were intermediate (Table 3). This finding corroborates the ideas of Eizaguirre et al. (2002), who suggested that the probiotic acted to lower the pH in the gut, subsequently improving the absorption of minerals such as calcium, iron and magnesium, due to an increased mineral solubility.

The SRBC, a nonpathogenic antigen, is classified as a thymus-dependent antigen that obviously needs the help of T lymphocytes to produce antibodies. Herbs that are rich in flavonoids such as thyme (Thymus vulgaris) extend the activity of vitamin C, act as antioxidants and may therefore enhance the immune function (Manach et al., 1996; Cook and Samman, 1996). In the present study, dietary treatments failed to have any significant (P>0.05) influence on antibody response to SRBC (Table 3). Similar results were obtained by previous researcher using thyme and antibiotic (Toghyani et al., 2010). Logambal et al. (2000) reported that a dose of 20 g leaf extract of Ocimum sanctum did not produce any significant change in the primary antibody response while evoked a significant (P<0.01) increase in the secondary antibody response. In the present study, the primary response to SRBC was measured after 7 days of injection. Thus, the lack of response to SRBC was probably due to the low level of thyme supplementation which failed to evoke the primary immune response.

Table 1. Ingredients and basal diet composition.

		Age, days
1-14	15-28	29-42
Ingredients, %
Corn	56.85	59.50	63.58
Soybean meal, 44% CP	39.00	34.30	30.00
Dicalcium phosphate	1.95	1.70	1.60
Oyster shell, 38% Ca	1.40	1.20	1.30
DL-Methionine	0.30	0.33	0.30
L-lysine	0.12	0.12	0.20
Common salt	0.33	0.30	0.30
NaHCO3	0.15	0.15	0.15
Micro ingredients premix^°	0.50	0.50	0.50
Sum	100	100	100
Calculated composition
Metabolizable energy, kcal/kg	2828	2940	3000
Crude protein, %	21.8	20.15	18.5
Calcium, %	1.05	0.90	0.90
Available phosphorus, %	0.50	0.45	0.42
Lysine, %	1.29	1.17	0.98
Total Sulfur Amino Acid, %	0.98	0.88	0.79
Sodium, %	0.18	0.18	0.18

°This premix supplied the following per kilogram of diet: vitamin A, 12,000 U; vitamin D₃, 5000 U; vitamin E, 75 U; vitamin K₃, 3 mg; thiamin, 3 mg; riboflavin, 8 mg; pantothenic acid, 13 mg; niacin, 55 mg; pyridoxine, 5 mg; folic acid, 2 mg; biotin, 0.2 mg; coline, 1600 mg; manganese, 120 mg; iron, 40 mg; copper, 16 mg; iodine, 1.25 mg; zinc, 100 mg; selenium, 0.3 mg.

Table 2. Effects of antibiotic, probiotic and thyme on broiler performance.

	C	AB	P	T1	T2	SEM	P value
Feed intake, g/bird
0-14 d	408.6	411.4	410.6	425.7	393.0	4.087	0.089
0-21 d	948.2	890.9	1007.1	1058.8	930.4	23.27	0.214
0-28 d	1971	1811	2054	1975	2106	79.80	0.273
0-35 d	3450	3094	3513	3292	3345	137.89	0.543
0-42 d	5684	4887	5418	4977	5590	257.67	0.370
Body weight, g
4 d	75.14	74.80	75.14	74.43	75.14	0.26	0.899
14 d	360	368	374	366	356	5.67	0.971
21 d	633	623	646	646	601	8.86	0.132
28 d	1236	1228	1231	1243	1185	11.63	0.566
35 d	1869	1873	1852	1882	1752	28	0.614
42 d	2222	2452	2405	2428	2421	45.93	0.556
Feed conversion ratio
0-14 d	1.135	1.118	1.098	1.163	1.104	0.019	0.435
0-21 d	1.498	1.430	1.559	1.639	1.549	0.028	0.569
0-28 d	1.595	1.475	1.669	1.589	1.777	0.054	0.535
0-35 d	1.846	1.652	1.897	1.749	1.909	0.087	0.340
0-42 d	2.558	1.993	2.253	2.050	2.309	0.126	0.087

C, control treatment; AB, diet supplemented with 2.5 mg/kg avilamycin; P, diet supplemented with 0.1 g/kg commercial probiotic; T1, diet supplemented with 5 g/kg ground thyme; T2, diet supplemented with 7.5 g/kg ground thyme.

Table 3. Effects of antibiotic, probiotic and thyme on blood parameters and SRBC responses at 21 and 35 days of age, respectively.

Treatment	Total protein, g/dL	Albumin, g/dL	Cholesterol, mg/dL	Triglyceride, mg/dL	Calcium, mg/dL	Phosphorus, mg/dL	SRBC
C	4.89^b	2.23^b	146.21^a	80.85^a	7.67^e	3.93^e	5.50
AB	5.27^b	2.52^b	133.51^b	70.66^b	8.30^d	4.81^d	4.82
P	6.52^a	3.14^a	102.70^d	45.41^e	13.98^a	7.29^a	5.11
T1	4.15^c	1.45^c	121.00^c	61.45^c	10.74^b	5.31^c	5.83
T2	2.58^d	1.09^d	114.25^c	52.81^d	9.92^c	6.14^b	5.67
SE	0.221	0.129	2.619	2.132	0.353	0.188	0.215
P value	0.001	0.001	0.001	0.001	0.001	0.001	0.565

C, control treatment; AB, diet supplemented with 2.5 mg/kg avilamycin; P, diet supplemented with 0.1 g/kg commercial probiotic; T1, diet supplemented with 5 g/kg ground thyme; T2, diet supplemented with 7.5 g/kg ground thyme.

^a,b,cMeans with different superscript letter within a column are significantly different (P<0.05).

Conclusions

A clear finding to emerge from this study is that probiotic and thyme supplementation at low levels (5 g/kg) in diets produced birds that were equivalent in performance to those fed supplementary avilamycin. Additionally, dietary supplementation with probiotics in particular, and, to intermediate extent ground thyme, had beneficial effects on blood biochemistry and mineral utilization in broilers.

However, these theoretical beneficial effects should be further studied with large samples in the condition of the commercial farms.