Effects of Different Levels of Hemp Seed (Cannabis Sativa L.) and Dextran Oligosaccharide on Growth Performance and Antibody Titer Response of Broiler Chickens

Abstract

A total of 384 Ross 308 male broilers were used in a randomised complete design for a period of six weeks. The experiment was conducted as a 4×2 factorial arrangement with 4 levels of hemp seed (HS) (0, 25, 50 and 75 g/kg) and 2 levels of dextran oligossacharide (DOS) (0 and 1 g/kg). Each of the 8 dietary treatments was fed to 4 replicate pens (12 birds/pen) from 1 to 42 d of age. The results indicated that diets containing 25 g/kg HS caused significant decrease in average daily feed intake and average daily gain (ADG), whereas total serum cholesterol, low and very low density lipoprotein, and TG levels was minimised at 75 g/kg of HS, but serum high density lipoprotein (HDL) was increased with increasing HS levels (P<0.01). Addition of 1 g/kg DOS diets compared with control diet, significantly increased ADG and HDL, and improved feed conversion ratio (P<0.05). Dietary DOS and HS had no significant effect on complete blood count, antibody production and relative weight of bursa and spleen in broilers (P>0.05). In conclusion, dietary HS and DOS can be used at their highest levels tested in the present study (75 g/kg of HS+1 g/kg of DOS) to decrease lipid levels of blood serum in broilers without any negative effect on performance.

Key Words:

• Antibody titer
• Broiler chicken
• Dextranoligosaccharide
• Hemp seed
• Performance

Introduction

Recently, new production concepts such as organic poultry production have intrigued nutrition scientists. Within organic production, there is a great need for knowledge about feed value, feeding strategies and feed utilisation (Jakobsen and Hermansen, 2001). Cannabis sativa L. is an annual, dioecious, green and leafy plant (Adams and Martin, 1996). It grows well at low temperature conditions where cultivating other oil seeds is too difficult (Karimi and Hayatghaibi, 2006). The most important products of Cannabis sativa are whole seed, hulled seed, seed meal, fibre, oil, marijuana and hashish (Adams and Martin, 1996; Callaway, 2004). The different products of Cannabis sativa have been used as traditional medicine in Asia, especially in Iran, to remedy different diseases such as nausea, headache, alopecia, diarrhea and inflammation. In ancient Iranian Avesta medicine, hashish was mixed with wine to deliver anesthesia. Whole hemp seed (HS) was shown to contain 18.0 MJ/kg metabolisable energy (ME) for birds (Hullar et al., 1999), 20% (Fortenbery and Bennet, 2004) to 24% protein (Hullar et al., 1999), 33% ether extract (EE) and 35% carbohydrate (mostly as fibre). Hemp seed protein is free of trypsin inhibitors and oligosaccharides found in soybeans that cause stomach upset and gas, and thus were used in traditional medicine to treat flatulence (Eriksson and Wall, 2012).

The great proportion of unsaturated fatty acids (typically 90%) and about 75 to 80% polyunsaturated fatty acids (PUFA), chiefly linoleic acid (LA) and alpha linolenic acid (ALA), in HS once made it desirable as a drying oil in industrial applications (Table 1; Callaway, 2004). Hemp seed is celebrated as the most unsaturated oil derived from the plant kingdom and has been dubbed as nature’s most perfectly balanced oil due to the fact that it contains the perfectly balanced 3:1 ratio of LA:ALA, determined to be the optimum requirement for long-term healthy human nutrition. Puthpongsiriporn and Scheideler (2005) reported that pullets fed diets containing 1:2 and 1:4, ALA:LA proportions had more antibody production against Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) without any negative effects on performance. In addition, HS is a good source of iron (1680 ppm) that could increase iron concentration in diets. However, due to the content of the intoxicating sticky resins produced most abundantly in the flowering tops of female plants before the seeds mature, delta9-tetrahydrocannabinol (Δ9-THC, hereafter simply THC) and less physically potent isomers such as Δ8-THC and tetrahydrocanabiverol (Small and Marcus, 2003), cultivating of HS was restricted or banned in more countries. There is limited research on hemp seed and dextran oligosaccharides as feed or feed additive for broilers, and thus there is a great need to investigate the role of hemp seed as a feed for broilers, as this study reflects.

For the past several decades, growth promoter feed additives have been included in poultry diets to promote growth, protect health and maximise the genetic potential of modern broiler, turkey and layer hybrids. Products such as prebiotics also have long been tested for their effects on the intestinal tract of healthy poultry and for safeguarding animal health status and performance (Kocher, 2005). Dextran is an oligosaccharide produced by growing Leuconostoc mesenteriodes bacteria on sucrose culture medium. Only lactic acid bacteria could digest this oligosaccharide because they have dextranase enzyme. Feeding dextran oligosaccharide (DOS) has been shown to reduce organ invasion by E. coli and Salmonella enteritidis, decrease cecal bacterial count (Fukuta et al., 1998), increase body weight (BW) in broilers (Bozkurt et al., 2008), thereby improving egg production performance and feed efficiency for egg number and output (Bozkurt et al., 2008). In addition, previous reports suggest that prebiotic supplementation resulted in significant improvement in antibody responses in broilers and layers (Raju and Devegowda, 2002; Shashidhara and Devegowda, 2003). Shafey et al. (2001) reported no such improvement in the antibody titers against IBDV and NDV in broilers fed prebiotics. The aim of this study was to evaluate the effect of different levels of dietary HS and DOS as prebiotic on average daily feed intake (ADFI), average daily gain (ADG), feed conversion ratio (FCR), NDV and IBV antibody responsiveness, complete blood count (CBC), serum cholesterol and triglycerides (TG) on broiler chicks.

Materials and methods

Experimental animals and diets

Three hundred and eighty four male broiler chicks (Ross 308) were obtained from a commercial hatchery and allocated to 32 floor pens (1×1 m) each containing 12 birds. Each pen had one feeder that was removable for weighing and two adjustable nipple drinkers. The average initial body weight of each group was similar (47 g±0.5; P>0.05). Pens were randomly assigned to the eight treatments (four pens for per treatment). All the pens were maintained under a uniform temperature and lighting control system during the entire period of study. The treatment structure consisted of a 4×2 factorial arrangement as a completely randomised design. A basal diet was formulated based on corn and soybean meal for the starter (0 to 14 d), grower (15 to 28 d) and finisher (29 to 42 d) periods. Basal diets were formulated to meet or exceed all nutrient recommendations published in the Ross rearing guideline (Table 2; Aviagen, 2014). Supplemental DOS (Meito Healthy Friend, MHF-Y; Meito Sangyo Co., Ltd., Tokyo, Japan) was substituted for equivalent amounts of corn in the basal diet. Diets were isoenergitic and isonitrogeneous and provided in mash form. The treatments were as follow: T₁, basal diet; T₂, basal diet+25 g/kg of HS; T₃, basal diet+50 g/kg of HS; T₄, basal diet+75 g/kg of HS; T₅, basal diet+1 g/kg of DOS; T₆, basal diet+25 g/kg of HS+1 g/kg of DOS; T₇, basal diet+50 g/kg of HS+1 g/kg of DOS; and T₈, basal diet+75 g/kg of HS+1 g/kg of DOS. The experiment lasted six wk. Birds had ad libitum access to feed and water during the experimental period and light was on continuously during the first day and 23 h/d afterward.

Vaccine programme

The following schedule describes the age and vaccine given: 1-old-day: IBV (Razi Inc., Karaj, Iran) attenuated live vaccine by eye drop; 8-old-day: NDV (Veterina Inc., Rakov Potok, Croatia) attenuated live vaccine by eye drop; 14-old-day: IBV (Intervet Inc., Summit, NJ, USA) attenuated live vaccine by eye drop.

Collection of samples and measurements

Chicken BW and feed intake (FI) by pen were recorded weekly. Average daily gain, ADFI and FCR were calculated for each phase. Dead birds were weighed and recorded daily. When calculating feed efficiency, the BW of the dead birds was taken into consideration.

At day 42, two randomly chosen birds per replicate (8 birds per treatment), representative of the mean body weight, were killed by cervical dislocation, and processed manually. The lymphoid organs (bursa and spleen) of the killed birds were removed and measured. Serum samples were separated from the clot by centrifugation at 3000 rpm for 15 min using bench top centrifuge (MSE Minor, London, UK). Serum samples were separated into sterile plain tubes and stored in a refrigerator for analyses.

Total antibody production specific for NDV vaccine or IBV vaccine was determined in serum by means of an ELISA using commercial ELISA test kits (IDEXX Laboratories Inc., Westbrook, ME, USA) (Jeffery et al., 1996). One microliter of serum was diluted 1:500 in 3.3, 5.5 -tetramethylbenzidine (TMB) diluents. Then 100 L of diluted sample was dispensed into each well of a 96-well plate coated with NDV, IBV, or IBDV antigen. After incubation for 30 min at room temperature, plates were washed three to five times with distilled water to remove non-adherent antibodies. One hundred microliters of goat antichicken: horseradish peroxidase conjugate was added to each well and incubated for 30 min at room temperature, and then plates were washed again three to five times with distilled water. One hundred microliters of TMB substrate solution was then added to each well, and after an incubation of 15 min at room temperature one µL of stop solution (0.125% hydrofluoric acid) was added to each well to stop the reaction. Antibody titers were measured as absorbance units at 650 nm by a microtiter plate reader using Xcheck software version 1.0.

Serum was used for TG, total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL) and very low density lipoprotein (VLDL) concentrations. Determinations were performed using an automated biochemical analyzer (Multianalyser Technicon RA-XT, Bayer do Bresil) with colorimetric methods, following the instructions of the manufacturer of the corresponding reagent kit (Zhongsheng Biochemical Co. Ltd., Beijing, China), and complete blood counts were counted manually.

Statistical analysis

Statistical analysis of results was performed by using the GLM procedure of SAS (SAS, 2006) according to the following general model:

Y_ijk=μ+H_i+D_j+HD_ij+e_(ijk)

where μ is the overall mean, H is the effect of hemp seed, D is the effect of DOS, HD is the interaction effect of dietary hemp seed by DOS and e is the residue error. Significant differences between means were compared by Duncan’s Multiple Range Test. When interaction effects were found to be significant with the F-test (P<0.05), the treatment means were separated by the least squares means function of SAS (SAS, 2006), with a confidence level of P<0.05.

Results and discussion

The results indicated that interaction effects between HS and DOS for ADG were significant (P<0.05) during the growth period of 1 to 21, but were not significant on ADFI, ADG and FCR during 22 to 42 and 1 to 42 d periods (Table 3). The inclusion of HS at 25 g/kg significantly decreased (P<0.01) ADFI and ADG, whereas ADG was maximised at 75 g/kg of HS. According to the current results, addition of DOS at 1 g/kg increased ADG during 22 to 42 and 1 to 42 d periods. All two-way interactions were non-significant except in the case of ADG during 1 to 21 d period (P<0.05). Throughout the experiment, there was only one case of mortality and it was in the control treatment. Because of this limited number of death cases no statistical analysis was performed. These effects may be due to orexigenic effects of tetrahydrocanabinol exist in HS (Mechoulam and Hanu, 2001). It is predicted that increasing in THC dosage, increased FI by increasing appetite at 50 and 75 g/kg HS in spite of the fact that dietary fibre increased. It is very well documented cannabinoids such as THC is involved in appetite, eating behaviour and body weight regulation. It is now confirmed that endocannabinoids as well as exocannabinoids, acting at brain CB1 cannabinoid receptors, stimulate appetite and ingestive behaviours, partly through interactions with more established orexigenic and anorexigenic signals. Karimi and Hayatghaibi (2006) showed that feeding HS to rats leads to increased body weight and serum protein. They reported that THC was an orexigenic compound and increases appetite as it is an antioxidant. These results are similar to our results. In agreement with our results, it is shown that supplementing diets with DOS had no significant effect on feed intake in broilers (Gibson and Roberfroid, 2008). Dietary inclusion of DOS (1 g/kg) increased WG at 22 to 42 d, 1 to 42 d and improved FC which was mainly due to beneficial effects of DOS on intestinal improvement and its microflora (Yalcinkaya et al., 2008). Yalcinkaya et al (2008) reported that prebiotics could increase nutrient absorption by increasing intestine length and microvillus compaction. Cannabis sativa has been shown to alleviate stress, improve immunity, suppress tumerous cells, having antimicrobial and antiviral activities (Novak et al., 2001). Moreover, it has also been reported for antiinflammatory, antipyretic, antiparasitic and insecticidal effects (Bishnupada et al., 1997). Combinations of these beneficial effects might have resulted in better performance of chicks given feed supplemented with 75 g/kg HS.

Effects of different levels of HS and DOS on broiler serum cholesterol are shown in Table 4. Serum HDL very significantly increased with dietary HS and increased with increasing HS levels (P<0.01). Concentration of LDL (P<0.01), VLDL (P<0.05), total cholesterol (P<0.01) and TG (P<0.05) of serum significantly decreased with increasing HS levels. McKenney and Sica (2007) reported that VLDL and TG level of blood serum will decrease with increasing unsaturated fatty acids, especially ALA concentration in diets of humans. The results of the present study are similar to previous studies. More and more consumers are strongly demanding safe and healthy by-products, which is the main reason why low-fat chickens are popular products in national markets. However, Zambón et al. (2000) reported that ALA enriched diets can increase HDL levels of human serum. Li et al. (2007) reported that supplementing diets with chito-oligosaccharide significantly increases HDL and LDL levels and decreases total cholesterol and TG levels of a broiler’s serum. Feeding diets supplemented with prebiotic decreases serum TG without any effect on lipid metabolism in a rat’s liver. Decreasing production of VLDL by liver leads to serum TG reduction in rats (Gibson and Roberfroid, 2008). Gibson and Roberfroid (2008) reported that prebiotics can reduce serum total cholesterol to one third due to decreasing production of lipoproteins, including high levels of TG. Thus the results of the present study are similar to previous studies. Effects of different levels of HS and DOS on CBC of broiler chicks are shown in Table 5. Dietary HS and DOS had no significant effect on CBC of broilers, although Hgb levels were increased numerically in DOS treated broilers. These results may be due to beneficial effects of DOS as prebiotic on iron digestion and absorption that is accepted by different researchers in different species (Delzenne et al., 1995; Yasuda et al., 2006).

The results of the current study showed that relative weight of lymphoid organs (bursa and spleen) and antibody production against IBV and NDV vaccines were not affected by dietary treatments. Due to high levels of ALA in HS, we predicted that HS could increase antibody production in chicks. Puthpongsiriporn and Scheideler (2005) showed that in chicks fed diets containing 1:2 and 1:4 proportion of ALA:LA antibody production of NDV were increased; however, other studies disagreed with their reports (Fritsche and Cassity, 1992; Sijben et al., 2001). In the present study, contrary to these reports, dietary HS did not significantly affect antibody production against IBV and NDV vaccines. Moreover, supplementing diets with DOS had no significant effect on IBV and NDV antibody responsiveness in broilers. Raju and Devegowda (2002) reported that prebiotics could increase antibody production in broilers and layers respectively. These contradictory results might be due to negative or side effects of THC on broilers’ immune responsiveness. Cannabinoides had two receptors CB1 and CB2 that have been found in all mammals, birds, fish, and reptiles (Begg et al., 2005). CB1 receptors are found primarily in the brain, and in both male and female reproductive systems. And CB2 receptors are found in the immune system, with the greatest density in the spleen which appears to be responsible for the antiinflammatory and possibly other therapeutic effects of cannabis (Núñez et al., 2004).

Table 1. Chemical composition and fatty acid profile of hemp seed.

Nutrient composition
ME, kcal/kg	4300
CP, g/kg	248
CF, g/kg	276
EE, g/kg	355
Ca, g/kg	1.5
P, g/kg	11.6
Lys, g/kg	10.3
Met, g/kg	5.8
Cys, g/kg	4.1
Arg, g/kg	31.0
Trp, g/kg	2.0
Typical fatty acid profiles, %
Palmitic acid	5
Stearic acid	2
Oleic acid	9
LA	56
ALA	22
GLA	4
PUFA	84
n6/n3 ratio	2.5

ME, metabolisable energy; CP, crude protein; CF, crude fibre; EE, ether extract; Ca, calcium; P, phosphorus; Lys, lysine; Met, methionine; Cys, cysteine; Arg, arginine; Trp, tryptophan; LA, linolenic acid; ALA, α-linolenic acid; GLA, γ-linolenic acid, PUFA, polyunsaturated fatty acid.

Table 2. Feed ingredients and nutrient composition of basal diets for experimental broiler chicks.

	Starter, days 0 to 14	Grower, days 15 to 28	Finisher, days 29 to 42
Ingredient, g/kg
Corn^°	556	562	599
Soybean meal (43% CP)	356	337	310
Fish meal	20	20	0
Plant oil	29	48	54
Oyster shell	14	12	12
Na-bicarbonate	1	1	2
DCP	12	10	13
Salt	2	2	2
Met	2	2	2
Lys	3	1	1
Premix^#	5	5	5
Calculated composition
ME, MJ/kg	12.66	13.19	13.40
CP, %	22.00	21.00	19.000
CF, %	3.90	3.78	3.67
Ca, %	1.04	0.91	0.84
P, %	0.50	0.45	0.44
Met, %	0.55	0.54	0.50
Lys, %	1.45	1.24	1.07
Met+Cys, %	0.85	0.83	0.75

CP, crude protein; DCP, dicalcium phosphate; Met, methionine; Lys, lysine; ME, methionine; CF, crude fibre; Ca, calcium; P, phosphorus; Cys, cysteine.

°For dextran oligosaccharides (DOS) groups, DOS was substituted for equivalent amounts of corn.

^#Provided (per kilogram of diet): vitamin A, 400 U; vitamin D₃, 250 U; vitamin E, 30 mg; vitamin C, 30 mg; vitamin K₃, 13 mg; vitamin B₁, 10 mg; vitamin B₂, 16 mg; vitamin B₆, 12 mg; vitamin B₁₂, 0.1 mg; Ca pantothenate, 60 mg; folic acid, 0.2 mg; nicotinic acid, 83 mg; choline, 105 mg; Co, 0.4 mg; Cu, 3.7 mg; I, 0.5 mg; Mn, 86 mg; Mg, 108 mg; Zn, 62 mg; Fe, 42 mg; Ca, 11 mg; Na, 390 mg; Cl, 671 mg; K, 78 mg; Met, 45 mg.

Table 3. Effect of different levels of hemp seed and dextran oligosaccharide on feed intake, weight gain, and feed conversion in broiler chickens from 1 to 42 day of age.

Treatments			ADFI, g			ADG, g			FCR, g/g
HS, g/kg	DOS, g/kg	1 to 21	22 to 42	1 to 42	1 to 21	22 to 42	1 to 42	1 to 21	22 to 42	1 to 42
0	0	39.3	138.0	88.7	25.5^a	69.1	47.3	1.54	2.01	1.88
25	0	35.0	129.9	82.4	20.9^d	60.3	40.6	1.68	2.17	2.04
50	0	36.3	131.3	83.8	20.9^d	70.6	45.7	1.77	1.87	184
75	0	38.6	140.7	89.7	24.1^abc	64.5	44.3	1.60	2.18	2.02
0	1	38.4	136.3	87.3	23.5^abcd	71.6	47.5	1.64	1.93	1.85
25	1	35.2	139.4	87.3	21.7^cd	69.7	45.7	1.62	2.00	1.91
50	1	36.0	138.6	87.3	22.6^bcd	70.1	46.3	1.60	1.98	1.89
75	1	38.4	141.1	89.8	25.3^a	78.5	51.9	1.5	1.81	1.73
SEM		0.88	13.50	2.21	0.82	2.08	1.23	0.03	0.03	0.02
Main effects
HS, g/kg
0		38.9^a	137.3	88.1	24.5^a	70.3	47.4^a	1.59	1.97	1.87
25		35.1^b	134.6	84.9	21.3^b	65.0	43.1^b	1.65	2.08	1.98
50		36.2^ab	135.0	85.6	21.7^b	70.3	46.0^ab	1.69	1.92	1.86
75		38.5^a	141.0	89.7	24.7^a	71.5	48.1^a	1.56	1.99	1.88
DOS, g/kg
0		37.3	135.0	86.2	22.8	66.1^b	44.5^b	1.65	2.06^a	1.94
1		37.0	138.9	88.0	23.2	72.5^a	47.9^a	1.60	1.92^b	1.85

ADFI, average daily feed intake; ADG, average daily gain; FCR, feed conversion ratio; HS, hemp seed; DOS, dextran oligosaccharide.

^a-dMean values within a column with no common superscript differ significantly from each other (P<0.05). Values represent 4 replicates of 12 birds per replicate for each of the 8 experiments.

Table 4. Effect of different levels of hemp seed and dextran oligosaccharide on serum cholesterol of broiler chickens (mg/dL).

Treatments		HDL	LDL	VLDL	Total cholesterol	TG
HS, g/kg	DOS, g/kg
0	0	81.0	81.3	7.9	170.2	39.4
25	0	82.7	79.1	7.6	169.5	38.0
50	0	84.7	74.0	7.4	166.2	37.2
75	0	85.5	66.9	7.1	159.5	35.6
0	1	84.0	80.2	7.6	171.7	37.8
25	1	84.2	78.0	7.3	169.7	37.3
50	1	85.2	75.7	8.3	168.0	36.4
75	1	85.2	69.8	7.1	162.2	35.9
SEM		0.22	0.75	0.06	0.73	0.29
Main effects
HS, g/kg
0		82.5^b	80.8^a	7.7^a	171.0^a	38.6^a
25		83.5^ab	78.6^a	7.6^ab	169.5^a	38.0^ab
50		84.7^ab	74.0^ab	7.4^ab	166.2^ab	37.2^ab
75		85.5^a	66.9^b	7.1^b	159.5^b	35.6^b
DOS, g/kg
0		83.5^b	75.4	7.5	166.4	37.6
1		84.7^a	75.9	7.4	167.9	36.9

HDL, high density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein; TG, triglycerides; HS, hemp seed; DOS, dextran oligosaccharide.

^a,bMean values within a column with no common superscript differ significantly from each other (P<0.05). Each value represents the mean of 8 observations (four replicates×2 birds/replicate).

Table 5. Effect of different levels of hemp seed and dextran oligosaccharide on complete blood count of broiler chickens (g/dL).

Treatments		Lym	Mono	Eos	Baso	Het	Band	RBC	Hct	Hgb
HS, g/kg	DOS, g/kg
0	0	55.75	0.75	0.50	0.25	42.75	0.00	3.50	31.50	10.5
25	0	54.75	0.75	0.75	0.00	43.50	0.25	3.67	33.00	11.00
50	0	54.50	0.75	1.00	0.25	43.25	0.25	3.72	33.50	11.17
75	0	54.50	0.50	0.75	0.00	44.00	0.25	3.72	33.50	11.91
0	1	54.35	0.75	1.25	0.25	43.00	0.50	3.67	33.00	11.00
25	1	54.00	0.50	0.75	0.00	44.50	0.25	3.83	33.50	11.50
50	1	54.00	0.00	1.25	0.25	44.50	0.00	3.81	34.25	11.41
75	1	55.75	0.50	0.75	0.00	42.50	0.25	3.74	33.50	11.17
SEM		0.39	0.14	0.17	0.07	0.35	0.07	0.03	0.30	0.10
Main effects
HS, g/kg
0		55.00	0.75	0.87	0.25	42.87	0.25	3.58	32.25	10.75
25		54.37	0.62	0.75	0.00	44.00	0.25	3.75	33.75	11.25
50		54.25	0.37	1.12	0.25	43.87	0.13	3.76	33.87	11.29
75		55.12	0.50	0.75	0.12	43.25	0.25	3.73	33.50	11.04
DOS, g/kg
0		54.87	0.69	0.75	0.12	43.37	0.19	3.65	32.87	10.89
1		54.50	0.44	1.00	0.19	43.62	0.25	3.76	33.81	11.27

Lym, lymphocyte; Mono, monocyte; Eos, eosinophile; Baso, basophile; Het, heterophile; Band, band cell; RBC, red blood cell (*106/μL); Hct, hematocryt; Hgb, hemoglobin; HS, hemp seed; DOS, dextran oligosaccharide. Each value represents the mean of 8 observations (four replicates×2 birds/replicate).

Conclusions

In this study, we found that dietary supplementation of HS can lower blood lipids in broilers. Dietary HS and DOS can be used at their highest levels tested in the present study (75 g/kg of HS+1 g/kg of DOS) to decrease lipid levels of blood serum in broilers. However, the broiler growth performance was negatively affected by dietary treatments at lower levels. To the best of our knowledge no similar work was found in literature, therefore, there is still a need to conduct more research in order to establish the suitability of such combinations to enhance satisfactory feed utilisation results that could be reflected on broiler growth performance.

Acknowledgments

this work was financially supported by the Urmia University, Iran. The authors wish to thank Dr. Arash Azarfar for his excellent technical assistance.