DOI: 10.1111/jpn.12115
ORIGINAL ARTICLE
Comparative effects of using black seed (Nigella sativa), cumin seed (Cuminum cyminum), probiotic or prebiotic on growth performance, blood haematology and serum biochemistry of broiler chicks K. Alimohamadi1, K. Taherpour1, H. A. Ghasemi2 and F. Fatahnia1 1 Department of Animal Science, Faculty of Agriculture, Ilam university, Ilam, Iran, and 2 Department of Animal Science, Faculty of Agriculture and Natural Resources, Arak University, Arak, Iran
Summary A 42-day trial was conducted to compare the effects of the following seven experimental diets, which varied in black seed, cumin seed, probiotic or prebiotic concentrations, on the broiler chicks: control (no additives), diet BS1 (4 g/kg black seed), diet BS2 (8 g/kg black seed), diet CS1 (4 g/kg cumin seed), diet CS2 (8 g/kg cumin seed), diet Pro (1 g/kg probiotic Primalacâ) and diet Pre (2 g/kg prebiotic Fermactoâ). A total of 420 1-day-old male broiler chicks, initially weighing an average of 43 g, were distributed into 28 floor pens at a stocking density of 15 birds per pen. At 28 day of age, the body weight in the birds fed diets BS2, CS2 and Pro was significantly higher than in the control group, but final body weight was not affected. Additionally, the birds fed diets BS2, Pro and Pre exhibited better feed conversion ratio than control birds from 0 to 42 day of age. Diets BS2, CS2 and Pro also statistically increased the relative weight of thymus and bursa of Fabricius, whereas only diet Pro decreased the abdominal fat percentage compared with control diet. Regarding the haematological parameters, feeding diet BS2 yielded a significant increase in red blood cell count, haemoglobin concentration and haematocrit percentage compared with control diet. Serum total cholesterol and low-density lipoprotein cholesterol levels in the birds fed diets BS2, Pro and Pre were also significantly lower than in the birds fed the control diet. Without exception, no diets affected feed intake, internal organs weights, carcass characteristics, antibody titres against Newcastle and influenza viruses and leucocyte subsets. In general, current study showed promising results regarding the use of spice additives as growth and health promoters, especially at higher levels of their incorporation in the diets, which were comparable to the probiotic- or prebiotic-containing diets. Keywords spice additives, probiotic, prebiotic, broilers performance, carcass traits, blood parameters Correspondence Dr. H. A. Ghasemi, Department of Animal Science, Faculty of Agriculture and Natural Resources, Arak University, Arak, 381568-8349, Iran. Tel: +98 861 2762087; Fax: +98 861 2761007; E-mail: [email protected] Received: 26 December 2012; accepted: 9 July 2013
The risk of bacteria becoming resistant to specific antibiotics and the residual effects of using antibiotic growth promoters (AGP) in poultry feed led to a ban on the use of antibiotics as growth promoters in animal production in European Union since January 2006 (Cross et al., 2007). With the removal of AGP from poultry diets and also increased concerns about food safety, general health risks and environmental contamination, the search for growth-promoting alternatives is necessary (Houshmand et al., 2011). In recent years, particular attention has been paid on the use of probiotics, prebiotics, and herbs and spices products as natural alternatives for AGP.
Probiotics and prebiotics are extensively used in poultry feed as an effective tool to eliminate or reduce pathogens or to promote adequate gastrointestinal health. Their important properties are as follows: the increase of bird’s resistance to diseases, the improvement of intestinal morphology and adjustment of immune function (Patterson and Burkholder, 2003). These additives are used not only as a growth promoter, but also they are able to reduce the absorption of bile acids from the intestine, resulting in decreased serum cholesterol level in broilers (Ooi and Liong, 2010). Probiotics are defined as ‘live microbial feed supplements, which beneficially affects the host animal by improving its intestinal microbial balance’ (Fuller, 1989). Prebiotics are indigestible food
Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
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Introduction
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ingredients, which can also have a direct effect on improving the gut health by nourishing and promoting the beneficial micro-organisms in the gut (Gibson and Roberfroid, 1995). However, inconsistencies exist in the literature regarding the effect of probiotics and prebiotics on growth performance. The reports vary from a beneficial effect (Samli et al., 2007; Yang et al., 2008; Awad et al., 2009), to no effect (Angel et al., 2005; Khodambashi-Emami et al., 2012). Among potential candidates, phytogenic additives (herbs and spices products) have also received increased attention as possible substitutions for AGP, as they have attained more acceptability among consumers as natural feed additives. Phytogenic additives are being used in animal nutrition as appetite, digestion stimulants, colourants and antioxidants, and also for prevention and treatment of pathological conditions (Hashemi and Davoodi, 2011). Previous studies have also demonstrated the positive effects of phytogenic additives on growth performance (AL-Beitawi et al., 2009), carcass characteristics (Al-Beitawi and El-Ghousein, 2008), immune system (KhodambashiEmami et al., 2012) and blood lipid profile (El-Dakhakhny et al., 2000). Black seed (Nigella sativa) is one of the oldest medicinal plants native to the Mediterranean region, south and south-west Asia, which belongs to family Ranunculacea. The main active ingredients of black seed include thymoquinone, dithymoquinone, thymohydroquinone, nigellone and thymol, which play important roles as pharmacologically active substances (Al-Saleh et al., 2006). Thymoquinone, representing 18.4–24% of the essential oil of the seeds, has antibacterial, antioxidant, anti-inflammatory and analgesic properties (Arslan et al., 2005). Cumin seed (Cuminum cyminum) is also an annual herbaceous plant of the Apiaceae family and possibly originated from the east Mediterranean to India (Lucchesi et al., 2004). Cumin contains 2.5–4% aromatic essential oils (cuminaldehyde and other aldehydes), and also a high concentration of antioxidant compounds, especially flavonoids and terpenes (El-Sawi and Mohamed, 2002). It is widely used in traditional herbal medicine and believed to possess particularly beneficial characteristics in promoting a healthier digestive system (Agarwal et al., 2010). AL-Beitawi et al. (2009) observed the beneficial influence of black seed on productive performance of broilers, but later in another study, Nasir and Grashorn (2010) failed to monitor any improvement in growth performance and carcass traits of broilers due to crushed black seeds. Also, the published reports regarding the effect of adding cumin seed to broiler diets on productive performance are inconsistent.
Four hundred and twenty-day-old male broiler chicks (Ross 308) were purchased from a local hatchery, weighed (43 0.35 g) and randomly allocated into one of seven treatments with four replicates of 15 chickens based on a completely randomized design. The diet was supplemented with black seed, cumin seed, probiotic and prebiotic alone on an as-fed basis. The concentrations of the administered supplements in the seven experimental diets were as follows: control diet (no supplement), diet BS1 (4 g/kg black seed), diet BS2 (8 g/kg black seed), diet CS1 (4 g/kg cumin seed), diet CS2 (8 g/kg cumin seed), diet Pro (1 g/kg probiotic) and diet Pre (2 g/kg prebiotic). In Table 1 are listed the feed ingredients and chemical composition of basal starter (1–10 days), grower (11–28 days) and finisher (29–42 days) diets. Diets formulated to meet the nutrient requirements of broilers according
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Al-Kassi (2010) reported a beneficial effect on body weight, feed efficiency and some carcass characteristics due to dietary cumin seed, whereas Pish-Jang (2011) did not observe a beneficial effect. As mentioned earlier, because of (i) inconsistencies in the results of different studies with probiotic, prebiotic and phytogenic additives on productive performance, (ii) lack of a research in which probiotic, prebiotics and spice additives were compared and (iii) the importance to find natural alternatives for AGP, this study was performed to evaluate the effect of black seed and cumin seed on male broilers growth performance, carcass characteristics, immune responses, serum biochemistry and blood haematology; and also to compare them with a commercial probiotic (Primalac) or a commercial prebiotic (Fermacto, Pet. Ag. Inc., Elgin, IL, USA). Materials and methods Supplements
The dried seeds of Nigella sativa and Cuminum cyminum were purchased from the local herbal market and powdered by grinding in a mixer grinder before adding to the experimental diets. The prebiotic (Fermacto) used in the present study was a carbohydrate belonged to mannan-oligosaccharides family and is attained by extraction of the outer cell wall of Saccharomyces cerevisiae. The commercial probiotic (Primalac, Star Labs, Clarksdale, MO, USA) was a combination of live microorganisms, including Lactobacillus acidophilus, Lactobacillus casei, Enterococcus faecium and Bifidobacterium bifidum. Animal and dietary treatments
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Spice additives, probiotic and prebiotic in broilers diets
Table 1 The feed ingredients and chemical composition of basal diets Ingredient g/kg of diet
1–10 day
11–28 day
29–42 day
Corn Soybean meal (44% CP) Fish meal Dicalcium phosphate Oyster shells Common salt DL-Methionine L-Lysine, HCL Vitamin pre-mix* Mineral pre-mix† Calculated composition ME, MJ/kg Crude protein, g/kg Ca, g/kg Non-phytate phosphorus, g/kg Lys, g/kg Met + Cys, g/kg
617.5 334.3 9.6 16.3 10.7 3.5 2.8 0.3 2.5 2.5
681.7 237.3 50.9 10.4 9.6 3.5 1.6 – 2.5 2.5
722.9 192.2 56.4 9.5 9.4 3.5 1.1 – 2.5 2.5
11.93 209.5 9 4.5 12.0 9.6
12.35 198 8.6 4.3 11.0 8.4
12.56 185 8.4 4.2 10.1 7.6
*Supplied per kilogram of diet: 6000 IU vitamin A, 800 IU vitamin D, 83 mg vitamin E, 2.2 mg vitamin K3, 2 mg vitamin B6, 8 mg vitamin B12, 10 mg nicotine amid, 0.3 mg folic acid, 20 mg D- biotin and 160 mg choline chloride. † Supplied per kilogram of diet: 32 mg Mn, 16 mg Fe, 24 mg Zn, 2 mg Cu, 800 lg I, 200 lg Co and 60 lg Se.
to the recommendations of Ross Broiler Manual (2002). All floor pens measured 1.3 9 2.5 m and had approximately 5 cm of built-up litter top dressed with new wood shaving. Each pen included a bell-type waterer and a tube feeder. Birds were vaccinated routinely against infectious bronchitis, Newcastle and Gambaro diseases, but no medication administered during the entire experimental period. All the chickens were maintained at a uniform temperature and lighting control system during the whole period of study. All procedures of the current study were approved by the Research Ethics Committee of Ilam University. Data collection and sampling
Body weights of the chicks were measured at 1, 10, 28 and 42 days and average daily feed intake (FI) was determined for different experimental periods. Feed conversion ratio (FCR) was calculated as FI:BWG at the end of each experimental period. At 42 days of age, two birds from each replicate were randomly chosen, slaughtered and abdominal fat pad, edible inner organs (heart, liver, gizzard), pancreas, small intestine, caecum and lymphoid organs (spleen, thymus and bursa of Fabricius) were collected, weighed and expressed as a percentage of live body weight. The Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
length of small intestine and caecum was also measured. Antibody response
At 18 and 28 days of age, two birds per replicate were randomly chosen and blood samples were collected from brachial vein and centrifuged to obtain serum. Antibody titres against Newcastle and influenza viruses were measured using ELISA kits (IDEXX Corb, Portland, ME, USA). Haematological parameters and blood leucocyte profiles
To study the effects of different dietary treatments on blood haematology at 42 days, blood samples were collected from eight birds in each treatment into EDTA-anticoagulant treated vials. The red blood cell (RBC) and white blood cell (WBC) counts were determined by a haemocytometer method using NattHerrick solution; haematocrit and haemoglobin values were measured by microhaematocrit and cyanmethaemoglobin methods respectively (Kececi et al., 1998). To determine blood leucocyte profiles, one hundred leucocytes per samples were counted by an optical microscope for heterophil to lymphocyte separation according to the protocol described by Lucas and Jamroz (1961) and then heterophil to lymphocyte ratio was calculated. Serum biochemical parameters
At 42 days of the experiment, 2 ml of blood was collected from the brachial vein from three birds of each pen (12 birds per treatment). Serum was isolated by centrifugation at 3000 9 g for 10 min. The serum concentrations of glucose, total protein, triglyceride (TG), total cholesterol (CHOL), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) in serum samples were analysed by an automatic biochemical analyzer (Clima, Ral. Co, Espain), following the instructions of the corresponding reagent kit (Pars Azmon Inc., Tehran, Iran). Statistical analysis
Data analysed in a randomized complete block design using the general linear model (GLM) procedure of SAS (SAS Institute, 2001). All results are presented as mean and the pooled SEM. Means were compared for significant (p ≤ 0.05) differences using the LSMEANS option of SAS. 3
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Results
Immunity
Growth performance and carcass characteristics
The additives used in the current study did not influence on overall antibody titres against Influenza and Newcastle viruses at 18 and 28 days of age (p > 0.05). However, as Table 3 shows, the weights of thymus and bursa of Fabricius in the birds fed diets BS2, CS2 or Pro were higher than those of the birds fed the control diet (p < 0.05).
The effects of dietary treatments on growth performance are shown in Table 2. At 28 days of age, diets BS2, CS2 and Pro increased body weight (p = 0.027). However, there were no significant differences in final body weight and average daily feed intake among any dietary treatments (p > 0.05). In contrast, the birds that were fed diets BS2, CS2 or Pro had significantly lower FCR than those fed the control diet from 11 to 28 days of age (p = 0.011); additionally, the chicks fed diets BS2, Pro and Pre exhibited significantly better FCR than chicks fed the control diet during the entire experimental period (p = 0.048). In the current study, we observed no influence of dietary treatments on carcass percentage, and relative weights of edible inner organs and pancreas (p > 0.05). Moreover, the relative weights and length of the small intestine and caecum were not statistically affected (p > 0.05). However, the abdominal fat percentage was significantly (p = 0.051) lower in the birds fed diet Pro (0.27) compared with control birds (0.37), whereas the birds fed the diets BS1 (0.33), BS2 (0.31), CS1 (0.29), CS2 (0.30) and Pre (0.29) were intermediate, and not significantly different from other groups.
Blood haematology
Effect of different dietary treatments on haematological parameters is given in Table 4. RBC counts, haemoglobin concentration and haematocrit percentage were significantly higher in the chicks fed diets BS2 compared with those fed the control diet (p < 0.05). However, WBC count, heterophil (H) and lymphocyte (L) counts and H/L ratio were not statistically affected by dietary treatments (p > 0.05). Serum biochemical parameters
Serum biochemical parameters at 42 days of age are presented in Table 5. Feeding diets BS2, Pro and Pre significantly decreased the concentrations of serum CHOL (p = 0.003) and LDL-C (p = 0.001) compared with the control diet. The serum TG levels were also
Table 2 The effect of different dietary additives on body weight gain (BWG), feed intake (g) and feed conversion ratio (FCR) in broiler chicks up to the age of 42 days Experimental diets* Item
Control
BS1
BS2
CS1
CS2
Pro
Pre
SEM
p value
Body weight (g) 10 days 28 days 42 days
152.2 855b 1989
153 892ab 2018
159.6 953a 2085
154.4 934ab 2056
164.3 950a 2068
162.7 981a 2139
155.9 936ab 2117
6.78 34.15 68.67
0.532 0.027 0.132
Daily feed intake (g) 1–10 days 11–28 days 29–42 days 1–42 days
17.1 69.6 153.3 85.0
17.0 68.9 150.7 83.8
17.6 68.9 147.7 82.9
17.1 71.7 150.9 85.1
18.5 69.1 149.8 84.0
18.2 71.9 150.5 85.3
17.5 70.2 152.3 85.0
1.24 5.97 10.15 9.25
0.329 0.763 0.825 0.748
Feed conversion ratio (g:g) 1–10 days 1.57 11–28 days 1.78a 29–42 days 1.89 1–42 days 1.84a
1.55 1.68ab 1.88 1.78ab
1.51 1.56b 1.83 1.71b
1.54 1.66ab 1.88 1.78ab
1.53 1.58b 1.88 1.74ab
1.52 1.58b 1.82 1.71b
1.56 1.62ab 1.80 1.72b
0.023 0.064 0.037 0.046
0.156 0.011 0.178 0.048
Values in the same row not sharing a common superscript differ significantly (p < 0.05). *Control diet, no supplement; diet BS1, 4 g/kg black seed; diet BS2, 8 g/kg black seed; diet CS1, 4 g/kg cumin seed; diet CS2, 8 g/kg cumin seed; diet Pro, 1 g/kg probiotic Primalacâ; and diet Pre, 2 g/kg prebiotic Fermactoâ.
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Spice additives, probiotic and prebiotic in broilers diets
Table 3 The effects of different dietary additives on relative weight of lymphoid organs in broiler chicks at 42 days of age Experimental diets* Item
Control
BS1
BS2
CS1
CS2
Pro
Pre
SEM
p value
Spleen Thymus Bursa of Fabricius
0.18 0.25b 0.08b
0.19 0.35ab 0.11ab
0.16 0.38a 0.17a
0.17 0.27ab 0.14ab
0.15 0.37a 0.18a
0.16 0.37a 0.18a
0.19 0.33ab 0.15ab
0.018 0.043 0.028
0.235 0.026 0.009
Values in the same row not sharing a common superscript differ significantly (p < 0.05). *Control diet, no supplement; diet BS1, 4 g/kg black seed; diet BS2, 8 g/kg black seed; diet CS1, 4 g/kg cumin seed; diet CS2, 8 g/kg cumin seed; diet Pro, 1 g/kg probiotic Primalacâ; and diet Pre, 2 g/kg prebiotic Fermactoâ.
Table 4 The effect of different dietary additives on various haematological parameters in broilers chicks at 42 days of age Experimental diets* Item
Control 6
BS1
b
RBC (910 /ll) WBC (9106/ll) Haemoglobin (mg/100 ml) Haematocrit (%) Leucocyte subsets Heterophil (H), % Lymphocyte (L), % H:L
BS2 ab
CS1 a
CS2 ab
Pro ab
Pre ab
ab
SEM
p value
1.96 22.2 11.5b 25b
2.10 22.1 12.6ab 26ab
2.25 22.7 13.1a 29a
2.03 23.1 11.9ab 27ab
2.17 22.9 12.2ab 27ab
2.16 22.6 12.8ab 28ab
2.13 22.9 12.6ab 27ab
0.11 0.94 0.63 1.27
0.036 0.877 0.046 0.033
17.3 82 0.21
16.8 84 0.20
13.5 85.5 0.16
17.5 82.3 0.21
11.8 81.3 0.14
10.8 83.5 0.13
16.8 81.3 0.21
2.48 4.19 0.038
0.231 0.567 0.145
RBC, red blood cell; WBC, white blood cell. Values in the same row not sharing a common superscript differ significantly (p < 0.05). *Control diet, no supplement; diet BS1, 4 g/kg black seed; diet BS2, 8 g/kg black seed; diet CS1, 4 g/kg cumin seed; diet CS2, 8 g/kg cumin seed; diet Pro, 1 g/kg probiotic Primalacâ; and diet Pre, 2 g/kg prebiotic Fermactoâ.
Table 5 The effect of different dietary additives on serum biochemical parameters in broilers chicks at 42 days of age Experimental diets* Item
Control
BS1
BS2
CS1
CS2
Pro
Pre
SEM
p value
Glucose (mg/dl) Total protein (g/dl) TG (mg/dl) CHOL (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl)
186 2.3 77.7a 147a 60.5 67.6a
212 2.6 63.8ab 130ab 61.6 57.8ab
217 3.3 66.4ab 119b 67.3 39.7b
213 2.5 66.5ab 135ab 66.7 56.6ab
217 2.6 58.2ab 128ab 72.4 48.6ab
200 2.9 50.3b 115b 75.6 32. 4b
207 2.9 61.7ab 121b 73.2 38.1b
11.8 0.4 6.4 8.9 6.1 9.5
0.367 0.567 0.023 0.003 0.087 0.001
CHOL, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol. Values in the same row not sharing a common superscript differ significantly (p < 0.05). *Control diet, no supplement; diet BS1, 4 g/kg black seed; diet BS2, 8 g/kg black seed; diet CS1, 4 g/kg cumin seed; diet CS2, 8 g/kg cumin seed; diet Pro, 1 g/kg probiotic Primalacâ; and diet Pre, 2 g/kg prebiotic Fermactoâ.
lower in the birds fed diets supplemented with probiotic compared with control birds (p = 0.023). There were no differences in serum glucose and total protein levels among dietary treatments (p > 0.05). However, The HDL-C levels were tended to be higher in the chicks fed diet supplemented with higher levels of spice seeds, probiotic or prebiotic versus the control diet (p = 0.087). Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
Discussion Growth performance
A higher body weight observed in the chicks fed 8 g/ kg black seed, 8 g/kg cumin seed or 1 g/kg probiotic at 28 days that was not reflected at slaughter age might be related to the fact that the birds decrease their nutritional requirements with age (NRC, 1994), and 5
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Spice additives, probiotic and prebiotic in broilers diets
also older birds have better developed visceral organs and digestive tracts (Lilja, 1983). It is also known that growth-promoting agents could not have more impact when the birds are kept under clean and disinfected conditions or fed with diets containing highly digestible ingredients (Lee et al., 2003). Therefore, lack of significant impact of all feed additives used in this study on final body weight at 42 day could be due to the ideal environmental conditions and/or the ideal composition of basal diet. Similar to our results, no positive response of body weight gain to phytogenic additives has been observed in other studies (Jonas et al., 2007; Nasir and Grashorn, 2010). In contrast, AL-Beitawi et al. (2009) reported an improved growth rate for broiler chicks fed diets supplemented with black seeds instead of bacitracin methylene disalicylate as AGP. Probably, the spice level applied in present study has not been such a level that would make a positive effect on growth rate, as there are reports of improved weight gain in broilers receiving diets supplemented with 15 g/kg black seeds (Al-Beitawi and El-Ghousein, 2008), or 10 and 15 g/kg cumin seeds (Al-Kassi, 2010), which are markedly higher levels in comparison with levels used in our study. The most efficient FCR in broiler chicks fed diets supplemented with 8 g/kg black seed, 1 g/kg probiotic and 2 g/kg prebiotic discloses that the impact of these growth-promoting substances on performance could be associated with a more efficient use of nutrient from the feed, which in turn results in an improved FCR. Some main ways in which probiotics and prebiotics can provide the feed efficiency benefit are improving intestinal microflora population and stimulating appetite as well as encouraging the immune system (Erdogan et al., 2010). The improvement of FCR by spices and their derivatives may be attributed to the stimulation of gastric and pancreatic digestive enzymes (Srinivasan, 2005), and/or modulation of microbial population by phytogenetic products (Windisch et al., 2008), which ultimately lead to more absorption of essential nutrients. Carcass traits
The results of this study are supported by other studies that similarly reported no observable change in the relative weights of internal organs, abdominal fat pad, carcass percentage, and the length of small intestine and caecum of broilers fed diets supplemented with probiotics, prebiotics (Falaki et al., 2011) and phytogenic additives (Toghyani et al., 2010). However, in the present study, the abdominal fat percentage was decreased by probiotic-supplemented diets, which is 6
in agreement with findings by Kalavathy et al. (2003). It is reported that probiotic supplementation can reduce hepatic mRNA expression of the lipogenic enzyme of acetyl-CoA carboxylase, the enzyme that catalyses limiting reaction in the biosynthesis of fatty acids, representing a decreased de novo lipogenesis (Axling et al., 2012). In addition, as indicated by Homma and Shinohara (2004), there is evidence that the reduction in body fat by probiotics may be due to more distribution of metabolic energy to maximize the heat produced by the temporary synthesis of muscle protein preceded than the fat storage in normal conditions. Immunity
The bursa of Fabricius and thymus are two specific lympho-epithelial organs in birds where the maturation and proliferation of B- and T -lymphocytes, respectively, take place. The positive effects of probiotics on these organs may be associated with increased rates of B and T cells turnover (Yakhkeshi et al., 2011), which could be a result of a promotion of B and T cells homeostasis in these organs. With respect to a higher relative weight of bursa of Fabricius and thymus recorded in chicks fed 8 g/kg black seed and 8 g/kg cumin seed, it is concluded that the active components of these spice seeds, which have antioxidant, antibacterial and anti-inflammatory activities (AlSaleh et al., 2006; Agarwal et al., 2010), could induce a positive effect on these organs. However, other immune-related parameters, including leucocyte profile and antibody titre against Newcastle and influenza viruses, neither positively nor negatively were affected. Because non-antibiotic feed supplements started to be used as growth promoters, researchers (Angel et al., 2005; Timmerman et al., 2006) working with broilers concluded that the presence of the main challenge in the field of environment and health is necessary to reveal the obvious effects of these products. This is while the present study was performed in the normal hygiene condition and using the healthy birds, which had no external stresses or challenges during the entire experimental period. Blood haematology
According to the obtained results concerning the haematology, it could be apparently deduced that the diets supplemented with 8 g/kg black seed have favourable effects on haematopoiesis. It increased RBC count, haemoglobin concentration and haematocrit percentage. Feeding rats with black seed has been Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
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reported to significantly reduce the haematological disorders induced by cadmium (Demir et al., 2006) and aflatoxin (Abdel-Wahhab and Aly, 2005). Also, Meral et al. (2004) findings demonstrated significant increase in RBC and WBC counts in alloxan-induced diabetic rabbits. The desirable effects of black seed on haematology can be due to the presence of pharmacologically active substances, particularly thymoquinone and thymohydoquinone, with strong antioxidant properties (Arslan et al., 2005). It can be therefore said that the higher RBC in the birds fed diet supplemented with 8 g/kg black seed could be attributed to the lowered lipid peroxide in erythrocyte membrane, resulting in a decreased susceptibility of erythrocyte to haemolysis. Serum biochemistry
Blood glucose reflects a balance of the amount of glucose absorbed from the small intestine, the glucose released by the liver into the blood and that going from the blood directly into the body’s cells. Serum protein also shows the overall condition of an organism and the alterations happening to it under the influence of stable internal and external factors. In our study, serum glucose and protein concentrations did not vary significantly among dietary treatments. Similarly, insignificant difference in blood glucose and total protein levels has been reported in broilers fed diets supplemented with probiotic (Capcarova et al., 2011), prebiotic (Ashayerizadeh et al., 2009) and spice products (Toghyani et al., 2010; Pish-Jang, 2011). However, the addition of 8 g/kg black seeds to diet resulted in a decrease in serum cholesterol and LDL-C, which could be because of the positive effects of phytogenic additives on lipid metabolism. Dietary administration of spice additives has been reported to have a hypocholesterolaemic effect in broilers, wherein these supplements decreased TG, CHOL and/or LDL-C levels and increased HDL-C levels (Pish-Jang, 2011; Yalcßın et al., 2012). In contrast, Toghyani et al. (2010) reported no significant effects of black seed and peppermint on serum lipids. The cholesterol-lowering effects of spice products are proposed to be associated with the reduction in the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), the ratecontrolling enzymes in cholesterol synthesis, in the liver by active components, especially essential oils, in spice seeds (El-Dakhakhny et al., 2000). The lower of de novo cholesterol synthesis also enhances the expression of low-density lipoprotein receptors on hepatocytes, leading to higher LDL uptake by the Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
Spice additives, probiotic and prebiotic in broilers diets
hepatocytes and ultimately lower the blood LDL-C levels (Fukushima and Nakano, 1996). Although, Al-Kassi (2010) reported that feeding broilers with 10 and 15 g/kg cumin seed reduced serum cholesterol and triglycerides compared with broilers fed control diet, our study failed to show any hypocholesterolaemic effects of cumin seed, possibly due to the low levels (4 and 8 g/kg of cumin seed compared with control). Moreover, It is a well-known fact that the absence or presence of cholesterolaemic effects of dietary components in an animal rely on various factors such as breed, gender, age and also on the composition of the feed. Probiotics and prebiotics have also been reported to possess hypocholesterolaemic and hypolipidaemic properties in poultry studies (Kalavathy et al., 2003; Velasco et al., 2010). The most important way that probiotics reduce serum cholesterol may be due to the fact that some probiotic bacteria, especially lactic acid-producing bacteria, may interfere with cholesterol absorption in the gut through deconjugating bile salts or by directly assimilating cholesterol (Ooi and Liong, 2010). The most likely mechanism by which prebiotics reduce serum cholesterol would likely be through binding bile acids, resulting in increased cholesterol removal by hepatic synthesis of new bile acid (Velasco et al., 2010). Furthermore, Robertfroid and Delzenne (1998) reported that these supplements reduce blood TG and LDL-C levels by decreasing lipogenesis in the liver. Conclusions In conclusion, considerable variations in growth performance and health benefits with the dietary use of spice additives are likely dependent on the supplement dose, feeding duration and type of supplement. The diet supplemented with 8 g/kg black seeds was advantageous over other diets containing spice additive and was similar with probiotic- or prebiotic-supplemented diets, with respect to feed efficiency. Moreover, feeding the higher levels of spice additives, as well as probiotic and prebiotic supplements, could improve lipid metabolism in broiler chickens. However, there is still a need to clarify the mechanisms of action for these natural herbs used in this study and also to assess the appropriate dose that should be safely used in the broiler diets. Acknowledgements The authors are grateful to Dr. Abdoreza Kamyab to supply prebiotic (Fermacto) from PetAg and probiotic (Primalac) from Starlabs in USA. 7
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References Abdel-Wahhab, M. A.; Aly, S. E., 2005: Antioxidant property of Nigella sativa (black cumion) and Syzygium aromaticum (clove) in rats during of latoxicosis. Journal of Applied Toxicology 25, 218–223. Agarwal, V.; Gupta, V.; Nepali, K.; Suri, O. P.; Dhar, K. L., 2010: Chemical and biological characteristics of Cuminum cyminum. Journal of Natura Conscientia 1, 148–156. Al-Beitawi, N.; El-Ghousein, S. S., 2008: Effect of feeding different levels of Nigella sativa seed (black cumin) on performance, blood constituents and carcass characteristics of broiler chicks. International Journal of Poultry Science 7, 715– 721. AL-Beitawi, N.A.; EL-Ghousein, S.S.; Nofal, A.H., 2009: Replacing bacitracin methylene disalicylate by crushed Nigella sativa seeds in broiler rations and its effects on growth, blood constituents and immunity. Livestock Science 125, 304–307. Al-Kassi, G. A. M., 2010: Effect of Feeding Cumin (Cuminum cyminum) on the Performance and Some Blood Traits of Broiler Chicks. Pakistan Journal of Nutrition 9, 72–75. Al-Saleh, I. A.; Billedo, G.; Inam, I. E., 2006: Level of selenium, DL-a-tocopgerol, DL-c-tocopherol, all trans retinol, thymoquinone and thymol in different brands of Nigella sativa seeds. Journal of Food Composition and Analysis 19, 167– 175. Angel, R.; Dalloul, R. A.; Doerr, J., 2005: Performance of broiler chickens fed diets supplemented with a direct-Fed microbial. Poultry Science 84, 1222–1231. Arslan, S. O.; Gelir, E.; Armutcu, F.; Coskun, O.; Gurel, A.; Sayan, H.; Celik, I. L., 2005: The protective effect of thymoquinone on ethanol-induced acute gastric damage in the rat. Nutrition Research 25, 673–680. Ashayerizadeh, O.; Dastar, B.; ShamsShargh, M.; Ashayerizadeh, A.; Mamooee, M., 2009: Influence of antibiotic, prebiotic and probiotic supplementation to diets on carcass characteristics, hematological indices and internal organ size of young broiler chickens. Journal of Animal and Veterinary Advances 8, 1772– 1776. Awad, W. A.; Ghareeb, K.; Abdel-Raheem, S.; Bohm, J., 2009: Effects of dietary inclusion of probiotic and synbiotic on
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growth performance, organ weights, and intestinal histomorphology of broiler chickens. Poultry Science 88, 49–56. Axling, U.; Olsson, C.; Xu, J.; Fernandez, C.; Larsson, S.; Str€ om, K.; Ahrn e, S.; Holm, C.; Molin, G.; Berger, K., 2012: Green tea powder and Lactobacillus plantarum affect gut microbiota, lipid metabolism and inflammation in highfat fed C57BL/6J mice. Nutrition & Metabolism 9, 105. doi:10.1186/ 1743-7075-9-105. Capcarova, M.; Weis, J.; Hrnc ar, C.; Koles arov a, A.; Petruska, P.; Kalafov a, A.; P al, G., 2011: Effect of probiotic supplementation on selected indices of energy profile and antioxidant status of chickens. Journal of Microbiology, Biotechnology and Food Sciences 1, 225–235. Cross, D. E.; Mcdevitt, R. M.; Hillman, K.; Acamovic, T., 2007: The effect of herbs and their associated essential oils on performance, dietary digestibility and gut microflora in chickens from 7 to 28 days of age. British Poultry Science 48, 496–506. Demir, H.; Kanter, M.; Coskun, O.; Uz, Y. H.; Koe, A.; Yildiz, A., 2006: Effect of black cumin (Nigella sativa) on heart rate, some hematological value, and pancreatic beta-cell damage in cadmium-treated rats. Biological Trace Element Research 110, 151–162. El-Dakhakhny, M.; Mady, N. I.; Halim, M. A., 2000: Nigella sativa oil protects against induced hepatotoxicity and improves serum lipid profile in rats. Arzneimittel-Forschung Drug Research 50, 832–836. El-Sawi, S. A.; Mohamed, M. A., 2002: Cumin herb as a new source of essential oils and its response to foliar spray with some microelements. Food Chemistry 77, 75–80. € Erdo gan, Z.; Erdo gan, S.; Aslantasß, O.; C ß elik, S., 2010: Effects of dietary supplementation of synbiotics and phytobiotics on performance, caecal coliform population and some oxidant/antioxidant parameters of broilers. Journal of Animal Physiology and Animal Nutrition 94, e40–e48. Falaki, M.; Shams-Shargh, M.; Dastar, B.; Zerehdaran, S., 2011: Effects of different levels of probiotic and prebiotic on performance and carcass characteristics of broiler chickens. Journal of Animal and Veterinary Advances 8, 2390–2395. Fukushima, M.; Nakano, M., 1996: Effects of a mixture of organisms, Lactobacillus
acidophilus or Streptococcus faecalis on cholesterol metabolism in rats fed on a fatand cholesterol- enriched diet. British Journal Nutrition 76, 857–867. Fuller, R., 1989: Probiotics in mans and animals. A review. Journal of Applied Bacteriology 66, 365–378. Gibson, G. R.; Roberfroid, M. B., 1995: Dietary modulation of human colonic microbiota: introducing the concept of prebiotics. Journal of Nutrition 125, 140– 142. Hashemi, S. R.; Davoodi, H., 2011: Herbal plants and their derivatives as growth and health promoters in animal nutrition. Veterinary Research Communications 35, 169–180. Homma, H.; Shinohara, T., 2004: Effects of probiotic Bacillus cereus toyoi on abdominal fat accumulation in the Japanese quail (Coturnix japonica). Animal Science Journal 75, 37–41. Houshmand, M.; Azhar, K.; Zulkifli, I.; Bejo, M. H.; Meimandipour, A.; Kamyab, A., 2011: Effects of non-antibiotic feed additives on performance, tibial dyschondroplasia incidence and tibia characteristics of broilers fed low-calcium diets. Journal of Animal Physiology and Animal Nutrition 95, 351–358. Jonas, D.; Skemaite, M.; Kirkilaite, G.; Vinauskiene, R.; Venskutonis, P. R., 2007: Antioxidant and antimicrobial properties of caraway (Carum carvi L.) and cumin (Cuminum cyminm) extracts. Veterinarija ir Zootechnika 40, 12–20. Kalavathy, R.; Abdullah, N.; Jalaludin, S.; Ho, Y. W., 2003: Effects of Lactobacillus cultures on growth performance, abdominal fat deposition, serum lipids and weight of organs of broiler chickens. British Poultry Science 44, 139–144. Kececi, O.; Oguz, H.; Kurtoglu, V.; Demet, € 1998: Effects of polyvinylpolypyrrO., olidone, synthetic zeolite and bentonite on serum biochemical and haematological characters of broiler chickens during aflatoxicosis. British Poultry Science 39, 452–458. Khodambashi-Emami, N.; Samie, A.; Rahmani, H. R.; Ruiz-Feria, C. A., 2012: The effect of peppermint essential oil and fructooligosaccharides, as alternatives to virginiamycin, on growth performance, digestibility, gut morphology and immune response of male broilers. Animal Feed Science and Technology 175, 57–64. Lee, K. W.; Everts, H.; Kappert, H. J.; Frehner, M.; Losa, R.; Beynen, A. C.,
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2003: Effects of dietary essential oil components on growth performance, digestive enzymes and lipid metabolism in female broiler chickens. British Poultry Science 44, 450–457. Lilja, C., 1983: Comparative study of postnatal growth and organ development in some species of birds. Growth 47, 317– 329. Lucas, A. M.; Jamroz, C., 1961: Atlas of Avian Hematology. Agriculture Monograph 25. USDA, Washington, DC. Lucchesi, M. E.; Chemat, F.; Smadja, J., 2004: An original solvent free microwave extraction of essential oils from spices. Flavour and Fragrance Journal 19, 134–138. Meral, I.; Donmez, N.; Baydas, B.; Belge, F.; Kanter, M., 2004: Effect of Nigella sativa L. on heart rate and some haematological values of alloxan induced diabetic rabbits. Scandinavian Journal of Laboratory Animal Science 31, 49–53. Nasir, Z.; Grashorn, M. A., 2010: Effects of Echinacea purpurea and Nigella sativa supplementation on broiler performance, carcass and meat quality. Journal of Animal and Feed Sciences 19, 94–110. NRC, 1994: National Research Council. Nutrient Requirements of Poultry. 9th rev. ed., National Academy Press, Washington, DC. Ooi, L. G.; Liong, M. T., 2010: Cholesterollowering effects of probiotics and prebiotics: a review of in vivo and in vitro findings. International Journal of Molecular Sciences 11, 2499–2522.
Spice additives, probiotic and prebiotic in broilers diets
Patterson, J. A.; Burkholder, K. M., 2003: Application of prebiotics and probiotics in poultry production. Poultry Science 82, 627–631. Pish-Jang, J., 2011: Comparison of effect of Cuminum cyminum and Probiotic on Performance and serum composition of broiler chickens. Annals of Biological Research 2, 630–634. Robertfroid, M. B.; Delzenne, N., 1998: Dietary fructans. Annual Review of Nutrition 18, 117–143. Ross Broiler Manual, 2002: Ross broiler management manual. Aviagen Ltd., Newbridge, Scottland. Samli, H. E.; Senkoylu, N.; Koc, F.; Kanter, M.; Agma, A., 2007: Effects of Enterococcus faecium and dried whey on broiler performance, gut histomorphology and microbiota. Archives of Animal Nutrition 61, 42–49. SAS Institute, 2001: SAS User’s Guide: Statistics. Version 8. SAS Institute, Cary, NC. Srinivasan, K., 2005: Spices as influencers of body metabolism: an overview of three decades of research. Food Research International 38, 77–86. Timmerman, H. M.; Veldman, A.; Van den Elsen, E.; Rombouts, F. M.; Beynen, A. C., 2006: Mortality and growth performance of broilers given drinking water supplemented with chicken-specific probiotics. Poultry Science 85, 1383–1388. Toghyani, M.; Toghyani, M.; Gheisari, A.; Ghalamkari, G.; Mohammadrezaei, M., 2010: Growth performance, serum biochemistry and blood hematology of broiler chicks fed different levels of black seed (Nigella sativa) and pepper-
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mint (Mentha piperita). Livestock Science 129, 173–178. Velasco, S.; Ortiz, L. T.; Alzueta, C.; Rebole, A.; Trevio, J.; Rodriguez, M. L., 2010: Effect of inulin supplementation and dietary fat source on performance, blood serum metabolites, liver lipids, abdominal fat deposition, and tissue fatty acid composition in broiler chickens. Poultry Science 89, 1651–1662. Windisch, W.; Schedle, K.; Plitzner, C.; Kroismayr, A., 2008: Use of phytogenic products as feed additives for swine and poultry. Journal of Animal Science 86, E140–E148. Yakhkeshi, S.; Rahimi, S.; Gharib-Naseri, K., 2011: The effects of comparison of herbal extracts, antibiotic, probiotic and organic acid on serum lipids, immune response, GIT microbial population, intestinal morphology and performance of broilers. Journal of Medicinal Plants 10, 80–95. glu, K.; DuYalcßın, S.; Yalcßın, S.; Uzuno € 2012: Effects of yum, H. M.; Eltan, O., dietary yeast autolysate (Saccharomyces cerevisiae) and black cumin seed (Nigella sativa L.) on performance, egg traits, some blood characteristics and antibody production of laying hens. Livestock Science 145, 13–20. Yang, Y.; Iji, P. A.; Kocher, A.; Mikkelsen, L. L.; Choct, M., 2008: Effects of dietary mannanoligosaccharide on growth performance, nutrient digestibility and gut development of broilers given different cereal-based diets. Journal of Animal Physiology and Animal Nutrition 92, 650–659.
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ORIGINAL ARTICLE
Comparative effects of using black seed (Nigella sativa), cumin seed (Cuminum cyminum), probiotic or prebiotic on growth performance, blood haematology and serum biochemistry of broiler chicks K. Alimohamadi1, K. Taherpour1, H. A. Ghasemi2 and F. Fatahnia1 1 Department of Animal Science, Faculty of Agriculture, Ilam university, Ilam, Iran, and 2 Department of Animal Science, Faculty of Agriculture and Natural Resources, Arak University, Arak, Iran
Summary A 42-day trial was conducted to compare the effects of the following seven experimental diets, which varied in black seed, cumin seed, probiotic or prebiotic concentrations, on the broiler chicks: control (no additives), diet BS1 (4 g/kg black seed), diet BS2 (8 g/kg black seed), diet CS1 (4 g/kg cumin seed), diet CS2 (8 g/kg cumin seed), diet Pro (1 g/kg probiotic Primalacâ) and diet Pre (2 g/kg prebiotic Fermactoâ). A total of 420 1-day-old male broiler chicks, initially weighing an average of 43 g, were distributed into 28 floor pens at a stocking density of 15 birds per pen. At 28 day of age, the body weight in the birds fed diets BS2, CS2 and Pro was significantly higher than in the control group, but final body weight was not affected. Additionally, the birds fed diets BS2, Pro and Pre exhibited better feed conversion ratio than control birds from 0 to 42 day of age. Diets BS2, CS2 and Pro also statistically increased the relative weight of thymus and bursa of Fabricius, whereas only diet Pro decreased the abdominal fat percentage compared with control diet. Regarding the haematological parameters, feeding diet BS2 yielded a significant increase in red blood cell count, haemoglobin concentration and haematocrit percentage compared with control diet. Serum total cholesterol and low-density lipoprotein cholesterol levels in the birds fed diets BS2, Pro and Pre were also significantly lower than in the birds fed the control diet. Without exception, no diets affected feed intake, internal organs weights, carcass characteristics, antibody titres against Newcastle and influenza viruses and leucocyte subsets. In general, current study showed promising results regarding the use of spice additives as growth and health promoters, especially at higher levels of their incorporation in the diets, which were comparable to the probiotic- or prebiotic-containing diets. Keywords spice additives, probiotic, prebiotic, broilers performance, carcass traits, blood parameters Correspondence Dr. H. A. Ghasemi, Department of Animal Science, Faculty of Agriculture and Natural Resources, Arak University, Arak, 381568-8349, Iran. Tel: +98 861 2762087; Fax: +98 861 2761007; E-mail: [email protected] Received: 26 December 2012; accepted: 9 July 2013
The risk of bacteria becoming resistant to specific antibiotics and the residual effects of using antibiotic growth promoters (AGP) in poultry feed led to a ban on the use of antibiotics as growth promoters in animal production in European Union since January 2006 (Cross et al., 2007). With the removal of AGP from poultry diets and also increased concerns about food safety, general health risks and environmental contamination, the search for growth-promoting alternatives is necessary (Houshmand et al., 2011). In recent years, particular attention has been paid on the use of probiotics, prebiotics, and herbs and spices products as natural alternatives for AGP.
Probiotics and prebiotics are extensively used in poultry feed as an effective tool to eliminate or reduce pathogens or to promote adequate gastrointestinal health. Their important properties are as follows: the increase of bird’s resistance to diseases, the improvement of intestinal morphology and adjustment of immune function (Patterson and Burkholder, 2003). These additives are used not only as a growth promoter, but also they are able to reduce the absorption of bile acids from the intestine, resulting in decreased serum cholesterol level in broilers (Ooi and Liong, 2010). Probiotics are defined as ‘live microbial feed supplements, which beneficially affects the host animal by improving its intestinal microbial balance’ (Fuller, 1989). Prebiotics are indigestible food
Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
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Introduction
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Spice additives, probiotic and prebiotic in broilers diets
ingredients, which can also have a direct effect on improving the gut health by nourishing and promoting the beneficial micro-organisms in the gut (Gibson and Roberfroid, 1995). However, inconsistencies exist in the literature regarding the effect of probiotics and prebiotics on growth performance. The reports vary from a beneficial effect (Samli et al., 2007; Yang et al., 2008; Awad et al., 2009), to no effect (Angel et al., 2005; Khodambashi-Emami et al., 2012). Among potential candidates, phytogenic additives (herbs and spices products) have also received increased attention as possible substitutions for AGP, as they have attained more acceptability among consumers as natural feed additives. Phytogenic additives are being used in animal nutrition as appetite, digestion stimulants, colourants and antioxidants, and also for prevention and treatment of pathological conditions (Hashemi and Davoodi, 2011). Previous studies have also demonstrated the positive effects of phytogenic additives on growth performance (AL-Beitawi et al., 2009), carcass characteristics (Al-Beitawi and El-Ghousein, 2008), immune system (KhodambashiEmami et al., 2012) and blood lipid profile (El-Dakhakhny et al., 2000). Black seed (Nigella sativa) is one of the oldest medicinal plants native to the Mediterranean region, south and south-west Asia, which belongs to family Ranunculacea. The main active ingredients of black seed include thymoquinone, dithymoquinone, thymohydroquinone, nigellone and thymol, which play important roles as pharmacologically active substances (Al-Saleh et al., 2006). Thymoquinone, representing 18.4–24% of the essential oil of the seeds, has antibacterial, antioxidant, anti-inflammatory and analgesic properties (Arslan et al., 2005). Cumin seed (Cuminum cyminum) is also an annual herbaceous plant of the Apiaceae family and possibly originated from the east Mediterranean to India (Lucchesi et al., 2004). Cumin contains 2.5–4% aromatic essential oils (cuminaldehyde and other aldehydes), and also a high concentration of antioxidant compounds, especially flavonoids and terpenes (El-Sawi and Mohamed, 2002). It is widely used in traditional herbal medicine and believed to possess particularly beneficial characteristics in promoting a healthier digestive system (Agarwal et al., 2010). AL-Beitawi et al. (2009) observed the beneficial influence of black seed on productive performance of broilers, but later in another study, Nasir and Grashorn (2010) failed to monitor any improvement in growth performance and carcass traits of broilers due to crushed black seeds. Also, the published reports regarding the effect of adding cumin seed to broiler diets on productive performance are inconsistent.
Four hundred and twenty-day-old male broiler chicks (Ross 308) were purchased from a local hatchery, weighed (43 0.35 g) and randomly allocated into one of seven treatments with four replicates of 15 chickens based on a completely randomized design. The diet was supplemented with black seed, cumin seed, probiotic and prebiotic alone on an as-fed basis. The concentrations of the administered supplements in the seven experimental diets were as follows: control diet (no supplement), diet BS1 (4 g/kg black seed), diet BS2 (8 g/kg black seed), diet CS1 (4 g/kg cumin seed), diet CS2 (8 g/kg cumin seed), diet Pro (1 g/kg probiotic) and diet Pre (2 g/kg prebiotic). In Table 1 are listed the feed ingredients and chemical composition of basal starter (1–10 days), grower (11–28 days) and finisher (29–42 days) diets. Diets formulated to meet the nutrient requirements of broilers according
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Al-Kassi (2010) reported a beneficial effect on body weight, feed efficiency and some carcass characteristics due to dietary cumin seed, whereas Pish-Jang (2011) did not observe a beneficial effect. As mentioned earlier, because of (i) inconsistencies in the results of different studies with probiotic, prebiotic and phytogenic additives on productive performance, (ii) lack of a research in which probiotic, prebiotics and spice additives were compared and (iii) the importance to find natural alternatives for AGP, this study was performed to evaluate the effect of black seed and cumin seed on male broilers growth performance, carcass characteristics, immune responses, serum biochemistry and blood haematology; and also to compare them with a commercial probiotic (Primalac) or a commercial prebiotic (Fermacto, Pet. Ag. Inc., Elgin, IL, USA). Materials and methods Supplements
The dried seeds of Nigella sativa and Cuminum cyminum were purchased from the local herbal market and powdered by grinding in a mixer grinder before adding to the experimental diets. The prebiotic (Fermacto) used in the present study was a carbohydrate belonged to mannan-oligosaccharides family and is attained by extraction of the outer cell wall of Saccharomyces cerevisiae. The commercial probiotic (Primalac, Star Labs, Clarksdale, MO, USA) was a combination of live microorganisms, including Lactobacillus acidophilus, Lactobacillus casei, Enterococcus faecium and Bifidobacterium bifidum. Animal and dietary treatments
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Table 1 The feed ingredients and chemical composition of basal diets Ingredient g/kg of diet
1–10 day
11–28 day
29–42 day
Corn Soybean meal (44% CP) Fish meal Dicalcium phosphate Oyster shells Common salt DL-Methionine L-Lysine, HCL Vitamin pre-mix* Mineral pre-mix† Calculated composition ME, MJ/kg Crude protein, g/kg Ca, g/kg Non-phytate phosphorus, g/kg Lys, g/kg Met + Cys, g/kg
617.5 334.3 9.6 16.3 10.7 3.5 2.8 0.3 2.5 2.5
681.7 237.3 50.9 10.4 9.6 3.5 1.6 – 2.5 2.5
722.9 192.2 56.4 9.5 9.4 3.5 1.1 – 2.5 2.5
11.93 209.5 9 4.5 12.0 9.6
12.35 198 8.6 4.3 11.0 8.4
12.56 185 8.4 4.2 10.1 7.6
*Supplied per kilogram of diet: 6000 IU vitamin A, 800 IU vitamin D, 83 mg vitamin E, 2.2 mg vitamin K3, 2 mg vitamin B6, 8 mg vitamin B12, 10 mg nicotine amid, 0.3 mg folic acid, 20 mg D- biotin and 160 mg choline chloride. † Supplied per kilogram of diet: 32 mg Mn, 16 mg Fe, 24 mg Zn, 2 mg Cu, 800 lg I, 200 lg Co and 60 lg Se.
to the recommendations of Ross Broiler Manual (2002). All floor pens measured 1.3 9 2.5 m and had approximately 5 cm of built-up litter top dressed with new wood shaving. Each pen included a bell-type waterer and a tube feeder. Birds were vaccinated routinely against infectious bronchitis, Newcastle and Gambaro diseases, but no medication administered during the entire experimental period. All the chickens were maintained at a uniform temperature and lighting control system during the whole period of study. All procedures of the current study were approved by the Research Ethics Committee of Ilam University. Data collection and sampling
Body weights of the chicks were measured at 1, 10, 28 and 42 days and average daily feed intake (FI) was determined for different experimental periods. Feed conversion ratio (FCR) was calculated as FI:BWG at the end of each experimental period. At 42 days of age, two birds from each replicate were randomly chosen, slaughtered and abdominal fat pad, edible inner organs (heart, liver, gizzard), pancreas, small intestine, caecum and lymphoid organs (spleen, thymus and bursa of Fabricius) were collected, weighed and expressed as a percentage of live body weight. The Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
length of small intestine and caecum was also measured. Antibody response
At 18 and 28 days of age, two birds per replicate were randomly chosen and blood samples were collected from brachial vein and centrifuged to obtain serum. Antibody titres against Newcastle and influenza viruses were measured using ELISA kits (IDEXX Corb, Portland, ME, USA). Haematological parameters and blood leucocyte profiles
To study the effects of different dietary treatments on blood haematology at 42 days, blood samples were collected from eight birds in each treatment into EDTA-anticoagulant treated vials. The red blood cell (RBC) and white blood cell (WBC) counts were determined by a haemocytometer method using NattHerrick solution; haematocrit and haemoglobin values were measured by microhaematocrit and cyanmethaemoglobin methods respectively (Kececi et al., 1998). To determine blood leucocyte profiles, one hundred leucocytes per samples were counted by an optical microscope for heterophil to lymphocyte separation according to the protocol described by Lucas and Jamroz (1961) and then heterophil to lymphocyte ratio was calculated. Serum biochemical parameters
At 42 days of the experiment, 2 ml of blood was collected from the brachial vein from three birds of each pen (12 birds per treatment). Serum was isolated by centrifugation at 3000 9 g for 10 min. The serum concentrations of glucose, total protein, triglyceride (TG), total cholesterol (CHOL), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) in serum samples were analysed by an automatic biochemical analyzer (Clima, Ral. Co, Espain), following the instructions of the corresponding reagent kit (Pars Azmon Inc., Tehran, Iran). Statistical analysis
Data analysed in a randomized complete block design using the general linear model (GLM) procedure of SAS (SAS Institute, 2001). All results are presented as mean and the pooled SEM. Means were compared for significant (p ≤ 0.05) differences using the LSMEANS option of SAS. 3
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Results
Immunity
Growth performance and carcass characteristics
The additives used in the current study did not influence on overall antibody titres against Influenza and Newcastle viruses at 18 and 28 days of age (p > 0.05). However, as Table 3 shows, the weights of thymus and bursa of Fabricius in the birds fed diets BS2, CS2 or Pro were higher than those of the birds fed the control diet (p < 0.05).
The effects of dietary treatments on growth performance are shown in Table 2. At 28 days of age, diets BS2, CS2 and Pro increased body weight (p = 0.027). However, there were no significant differences in final body weight and average daily feed intake among any dietary treatments (p > 0.05). In contrast, the birds that were fed diets BS2, CS2 or Pro had significantly lower FCR than those fed the control diet from 11 to 28 days of age (p = 0.011); additionally, the chicks fed diets BS2, Pro and Pre exhibited significantly better FCR than chicks fed the control diet during the entire experimental period (p = 0.048). In the current study, we observed no influence of dietary treatments on carcass percentage, and relative weights of edible inner organs and pancreas (p > 0.05). Moreover, the relative weights and length of the small intestine and caecum were not statistically affected (p > 0.05). However, the abdominal fat percentage was significantly (p = 0.051) lower in the birds fed diet Pro (0.27) compared with control birds (0.37), whereas the birds fed the diets BS1 (0.33), BS2 (0.31), CS1 (0.29), CS2 (0.30) and Pre (0.29) were intermediate, and not significantly different from other groups.
Blood haematology
Effect of different dietary treatments on haematological parameters is given in Table 4. RBC counts, haemoglobin concentration and haematocrit percentage were significantly higher in the chicks fed diets BS2 compared with those fed the control diet (p < 0.05). However, WBC count, heterophil (H) and lymphocyte (L) counts and H/L ratio were not statistically affected by dietary treatments (p > 0.05). Serum biochemical parameters
Serum biochemical parameters at 42 days of age are presented in Table 5. Feeding diets BS2, Pro and Pre significantly decreased the concentrations of serum CHOL (p = 0.003) and LDL-C (p = 0.001) compared with the control diet. The serum TG levels were also
Table 2 The effect of different dietary additives on body weight gain (BWG), feed intake (g) and feed conversion ratio (FCR) in broiler chicks up to the age of 42 days Experimental diets* Item
Control
BS1
BS2
CS1
CS2
Pro
Pre
SEM
p value
Body weight (g) 10 days 28 days 42 days
152.2 855b 1989
153 892ab 2018
159.6 953a 2085
154.4 934ab 2056
164.3 950a 2068
162.7 981a 2139
155.9 936ab 2117
6.78 34.15 68.67
0.532 0.027 0.132
Daily feed intake (g) 1–10 days 11–28 days 29–42 days 1–42 days
17.1 69.6 153.3 85.0
17.0 68.9 150.7 83.8
17.6 68.9 147.7 82.9
17.1 71.7 150.9 85.1
18.5 69.1 149.8 84.0
18.2 71.9 150.5 85.3
17.5 70.2 152.3 85.0
1.24 5.97 10.15 9.25
0.329 0.763 0.825 0.748
Feed conversion ratio (g:g) 1–10 days 1.57 11–28 days 1.78a 29–42 days 1.89 1–42 days 1.84a
1.55 1.68ab 1.88 1.78ab
1.51 1.56b 1.83 1.71b
1.54 1.66ab 1.88 1.78ab
1.53 1.58b 1.88 1.74ab
1.52 1.58b 1.82 1.71b
1.56 1.62ab 1.80 1.72b
0.023 0.064 0.037 0.046
0.156 0.011 0.178 0.048
Values in the same row not sharing a common superscript differ significantly (p < 0.05). *Control diet, no supplement; diet BS1, 4 g/kg black seed; diet BS2, 8 g/kg black seed; diet CS1, 4 g/kg cumin seed; diet CS2, 8 g/kg cumin seed; diet Pro, 1 g/kg probiotic Primalacâ; and diet Pre, 2 g/kg prebiotic Fermactoâ.
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Table 3 The effects of different dietary additives on relative weight of lymphoid organs in broiler chicks at 42 days of age Experimental diets* Item
Control
BS1
BS2
CS1
CS2
Pro
Pre
SEM
p value
Spleen Thymus Bursa of Fabricius
0.18 0.25b 0.08b
0.19 0.35ab 0.11ab
0.16 0.38a 0.17a
0.17 0.27ab 0.14ab
0.15 0.37a 0.18a
0.16 0.37a 0.18a
0.19 0.33ab 0.15ab
0.018 0.043 0.028
0.235 0.026 0.009
Values in the same row not sharing a common superscript differ significantly (p < 0.05). *Control diet, no supplement; diet BS1, 4 g/kg black seed; diet BS2, 8 g/kg black seed; diet CS1, 4 g/kg cumin seed; diet CS2, 8 g/kg cumin seed; diet Pro, 1 g/kg probiotic Primalacâ; and diet Pre, 2 g/kg prebiotic Fermactoâ.
Table 4 The effect of different dietary additives on various haematological parameters in broilers chicks at 42 days of age Experimental diets* Item
Control 6
BS1
b
RBC (910 /ll) WBC (9106/ll) Haemoglobin (mg/100 ml) Haematocrit (%) Leucocyte subsets Heterophil (H), % Lymphocyte (L), % H:L
BS2 ab
CS1 a
CS2 ab
Pro ab
Pre ab
ab
SEM
p value
1.96 22.2 11.5b 25b
2.10 22.1 12.6ab 26ab
2.25 22.7 13.1a 29a
2.03 23.1 11.9ab 27ab
2.17 22.9 12.2ab 27ab
2.16 22.6 12.8ab 28ab
2.13 22.9 12.6ab 27ab
0.11 0.94 0.63 1.27
0.036 0.877 0.046 0.033
17.3 82 0.21
16.8 84 0.20
13.5 85.5 0.16
17.5 82.3 0.21
11.8 81.3 0.14
10.8 83.5 0.13
16.8 81.3 0.21
2.48 4.19 0.038
0.231 0.567 0.145
RBC, red blood cell; WBC, white blood cell. Values in the same row not sharing a common superscript differ significantly (p < 0.05). *Control diet, no supplement; diet BS1, 4 g/kg black seed; diet BS2, 8 g/kg black seed; diet CS1, 4 g/kg cumin seed; diet CS2, 8 g/kg cumin seed; diet Pro, 1 g/kg probiotic Primalacâ; and diet Pre, 2 g/kg prebiotic Fermactoâ.
Table 5 The effect of different dietary additives on serum biochemical parameters in broilers chicks at 42 days of age Experimental diets* Item
Control
BS1
BS2
CS1
CS2
Pro
Pre
SEM
p value
Glucose (mg/dl) Total protein (g/dl) TG (mg/dl) CHOL (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl)
186 2.3 77.7a 147a 60.5 67.6a
212 2.6 63.8ab 130ab 61.6 57.8ab
217 3.3 66.4ab 119b 67.3 39.7b
213 2.5 66.5ab 135ab 66.7 56.6ab
217 2.6 58.2ab 128ab 72.4 48.6ab
200 2.9 50.3b 115b 75.6 32. 4b
207 2.9 61.7ab 121b 73.2 38.1b
11.8 0.4 6.4 8.9 6.1 9.5
0.367 0.567 0.023 0.003 0.087 0.001
CHOL, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol. Values in the same row not sharing a common superscript differ significantly (p < 0.05). *Control diet, no supplement; diet BS1, 4 g/kg black seed; diet BS2, 8 g/kg black seed; diet CS1, 4 g/kg cumin seed; diet CS2, 8 g/kg cumin seed; diet Pro, 1 g/kg probiotic Primalacâ; and diet Pre, 2 g/kg prebiotic Fermactoâ.
lower in the birds fed diets supplemented with probiotic compared with control birds (p = 0.023). There were no differences in serum glucose and total protein levels among dietary treatments (p > 0.05). However, The HDL-C levels were tended to be higher in the chicks fed diet supplemented with higher levels of spice seeds, probiotic or prebiotic versus the control diet (p = 0.087). Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
Discussion Growth performance
A higher body weight observed in the chicks fed 8 g/ kg black seed, 8 g/kg cumin seed or 1 g/kg probiotic at 28 days that was not reflected at slaughter age might be related to the fact that the birds decrease their nutritional requirements with age (NRC, 1994), and 5
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Spice additives, probiotic and prebiotic in broilers diets
also older birds have better developed visceral organs and digestive tracts (Lilja, 1983). It is also known that growth-promoting agents could not have more impact when the birds are kept under clean and disinfected conditions or fed with diets containing highly digestible ingredients (Lee et al., 2003). Therefore, lack of significant impact of all feed additives used in this study on final body weight at 42 day could be due to the ideal environmental conditions and/or the ideal composition of basal diet. Similar to our results, no positive response of body weight gain to phytogenic additives has been observed in other studies (Jonas et al., 2007; Nasir and Grashorn, 2010). In contrast, AL-Beitawi et al. (2009) reported an improved growth rate for broiler chicks fed diets supplemented with black seeds instead of bacitracin methylene disalicylate as AGP. Probably, the spice level applied in present study has not been such a level that would make a positive effect on growth rate, as there are reports of improved weight gain in broilers receiving diets supplemented with 15 g/kg black seeds (Al-Beitawi and El-Ghousein, 2008), or 10 and 15 g/kg cumin seeds (Al-Kassi, 2010), which are markedly higher levels in comparison with levels used in our study. The most efficient FCR in broiler chicks fed diets supplemented with 8 g/kg black seed, 1 g/kg probiotic and 2 g/kg prebiotic discloses that the impact of these growth-promoting substances on performance could be associated with a more efficient use of nutrient from the feed, which in turn results in an improved FCR. Some main ways in which probiotics and prebiotics can provide the feed efficiency benefit are improving intestinal microflora population and stimulating appetite as well as encouraging the immune system (Erdogan et al., 2010). The improvement of FCR by spices and their derivatives may be attributed to the stimulation of gastric and pancreatic digestive enzymes (Srinivasan, 2005), and/or modulation of microbial population by phytogenetic products (Windisch et al., 2008), which ultimately lead to more absorption of essential nutrients. Carcass traits
The results of this study are supported by other studies that similarly reported no observable change in the relative weights of internal organs, abdominal fat pad, carcass percentage, and the length of small intestine and caecum of broilers fed diets supplemented with probiotics, prebiotics (Falaki et al., 2011) and phytogenic additives (Toghyani et al., 2010). However, in the present study, the abdominal fat percentage was decreased by probiotic-supplemented diets, which is 6
in agreement with findings by Kalavathy et al. (2003). It is reported that probiotic supplementation can reduce hepatic mRNA expression of the lipogenic enzyme of acetyl-CoA carboxylase, the enzyme that catalyses limiting reaction in the biosynthesis of fatty acids, representing a decreased de novo lipogenesis (Axling et al., 2012). In addition, as indicated by Homma and Shinohara (2004), there is evidence that the reduction in body fat by probiotics may be due to more distribution of metabolic energy to maximize the heat produced by the temporary synthesis of muscle protein preceded than the fat storage in normal conditions. Immunity
The bursa of Fabricius and thymus are two specific lympho-epithelial organs in birds where the maturation and proliferation of B- and T -lymphocytes, respectively, take place. The positive effects of probiotics on these organs may be associated with increased rates of B and T cells turnover (Yakhkeshi et al., 2011), which could be a result of a promotion of B and T cells homeostasis in these organs. With respect to a higher relative weight of bursa of Fabricius and thymus recorded in chicks fed 8 g/kg black seed and 8 g/kg cumin seed, it is concluded that the active components of these spice seeds, which have antioxidant, antibacterial and anti-inflammatory activities (AlSaleh et al., 2006; Agarwal et al., 2010), could induce a positive effect on these organs. However, other immune-related parameters, including leucocyte profile and antibody titre against Newcastle and influenza viruses, neither positively nor negatively were affected. Because non-antibiotic feed supplements started to be used as growth promoters, researchers (Angel et al., 2005; Timmerman et al., 2006) working with broilers concluded that the presence of the main challenge in the field of environment and health is necessary to reveal the obvious effects of these products. This is while the present study was performed in the normal hygiene condition and using the healthy birds, which had no external stresses or challenges during the entire experimental period. Blood haematology
According to the obtained results concerning the haematology, it could be apparently deduced that the diets supplemented with 8 g/kg black seed have favourable effects on haematopoiesis. It increased RBC count, haemoglobin concentration and haematocrit percentage. Feeding rats with black seed has been Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
K. Alimohamadi et al.
reported to significantly reduce the haematological disorders induced by cadmium (Demir et al., 2006) and aflatoxin (Abdel-Wahhab and Aly, 2005). Also, Meral et al. (2004) findings demonstrated significant increase in RBC and WBC counts in alloxan-induced diabetic rabbits. The desirable effects of black seed on haematology can be due to the presence of pharmacologically active substances, particularly thymoquinone and thymohydoquinone, with strong antioxidant properties (Arslan et al., 2005). It can be therefore said that the higher RBC in the birds fed diet supplemented with 8 g/kg black seed could be attributed to the lowered lipid peroxide in erythrocyte membrane, resulting in a decreased susceptibility of erythrocyte to haemolysis. Serum biochemistry
Blood glucose reflects a balance of the amount of glucose absorbed from the small intestine, the glucose released by the liver into the blood and that going from the blood directly into the body’s cells. Serum protein also shows the overall condition of an organism and the alterations happening to it under the influence of stable internal and external factors. In our study, serum glucose and protein concentrations did not vary significantly among dietary treatments. Similarly, insignificant difference in blood glucose and total protein levels has been reported in broilers fed diets supplemented with probiotic (Capcarova et al., 2011), prebiotic (Ashayerizadeh et al., 2009) and spice products (Toghyani et al., 2010; Pish-Jang, 2011). However, the addition of 8 g/kg black seeds to diet resulted in a decrease in serum cholesterol and LDL-C, which could be because of the positive effects of phytogenic additives on lipid metabolism. Dietary administration of spice additives has been reported to have a hypocholesterolaemic effect in broilers, wherein these supplements decreased TG, CHOL and/or LDL-C levels and increased HDL-C levels (Pish-Jang, 2011; Yalcßın et al., 2012). In contrast, Toghyani et al. (2010) reported no significant effects of black seed and peppermint on serum lipids. The cholesterol-lowering effects of spice products are proposed to be associated with the reduction in the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), the ratecontrolling enzymes in cholesterol synthesis, in the liver by active components, especially essential oils, in spice seeds (El-Dakhakhny et al., 2000). The lower of de novo cholesterol synthesis also enhances the expression of low-density lipoprotein receptors on hepatocytes, leading to higher LDL uptake by the Journal of Animal Physiology and Animal Nutrition © 2013 Blackwell Verlag GmbH
Spice additives, probiotic and prebiotic in broilers diets
hepatocytes and ultimately lower the blood LDL-C levels (Fukushima and Nakano, 1996). Although, Al-Kassi (2010) reported that feeding broilers with 10 and 15 g/kg cumin seed reduced serum cholesterol and triglycerides compared with broilers fed control diet, our study failed to show any hypocholesterolaemic effects of cumin seed, possibly due to the low levels (4 and 8 g/kg of cumin seed compared with control). Moreover, It is a well-known fact that the absence or presence of cholesterolaemic effects of dietary components in an animal rely on various factors such as breed, gender, age and also on the composition of the feed. Probiotics and prebiotics have also been reported to possess hypocholesterolaemic and hypolipidaemic properties in poultry studies (Kalavathy et al., 2003; Velasco et al., 2010). The most important way that probiotics reduce serum cholesterol may be due to the fact that some probiotic bacteria, especially lactic acid-producing bacteria, may interfere with cholesterol absorption in the gut through deconjugating bile salts or by directly assimilating cholesterol (Ooi and Liong, 2010). The most likely mechanism by which prebiotics reduce serum cholesterol would likely be through binding bile acids, resulting in increased cholesterol removal by hepatic synthesis of new bile acid (Velasco et al., 2010). Furthermore, Robertfroid and Delzenne (1998) reported that these supplements reduce blood TG and LDL-C levels by decreasing lipogenesis in the liver. Conclusions In conclusion, considerable variations in growth performance and health benefits with the dietary use of spice additives are likely dependent on the supplement dose, feeding duration and type of supplement. The diet supplemented with 8 g/kg black seeds was advantageous over other diets containing spice additive and was similar with probiotic- or prebiotic-supplemented diets, with respect to feed efficiency. Moreover, feeding the higher levels of spice additives, as well as probiotic and prebiotic supplements, could improve lipid metabolism in broiler chickens. However, there is still a need to clarify the mechanisms of action for these natural herbs used in this study and also to assess the appropriate dose that should be safely used in the broiler diets. Acknowledgements The authors are grateful to Dr. Abdoreza Kamyab to supply prebiotic (Fermacto) from PetAg and probiotic (Primalac) from Starlabs in USA. 7
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