Dietary effects of buckwheat ( Fagopyrum esculentum ) and black cumin ( Nigella sativa ) seed on growth performance , serum lipid profile and intestinal microflora of broiler chicks

M.S. Islam, M.N. Siddiqui, M.A. Sayed, M. Tahjib-Ul-Arif, M.A. Islam & M.A. Hossain 1 Department of Biochemistry and Molecular Biology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh 2 Department of Biochemistry and Molecular Biology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh 3 The United Graduate School of Agricultural Sciences, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550 Japan 4 Department of Biochemistry and Molecular Biology, Hajee Mohammad Danesh Science & Technology University, Dinajpur-5200, Bangladesh 5 Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh


Introduction
Feed additive antibiotics reduce the cost and improve the quantity of food production through more efficient use of natural resources, which is critical to meet the escalating nutritional needs of a growing world human population.Although good results are obtained with these substances, the risk of the development of direct antibiotic resistance of pathogens in the species receiving the feed, as well as indirect resistance to similar antibiotics used in human medicine as the result of food chain residues, led to the ban of all subtherapeutic levels of growth-promoting antibiotics by the European Union (Council of the European Union, 1970) and many other countries, including Bangladesh.As this may negatively affect the profitability of poultry farming, alternative substances and strategies for growth promotion and disease prevention are being investigated, among which phytogenic and herbal products have received increased attention, since they have acquired acceptability among consumers as natural additives (Toghyani et al., 2010).In previous studies the authors found that phytogenic feed additives of plant origin normally proved safe, healthy, ecofriendly, cost effective, not directly associated with problems and less hazardous than synthetic feed additives in poultry production (Islam et al., 2011;Rahman et al., 2013;Islam et al., 2014;Sayed et al., 2015;Siddiqui et al., 2015).
One of the alternatives to synthetic antibiotic feed additives in poultry feed could be the seeds of the much-acclaimed herbal medicinal plant, black cumin (Nigella sativa L.).Black cumin is an annual herb of the Mediterranean region and belongs to the family Ranunculaceae.The main active components of black cumin seed include thymoquinone, thymohydroquinone, dithymoquinone, thymol and carvacrol, which are important pharmacologically active substances (Nasir et al., 2005;Al-Saleh et al., 2006).The seeds have been reported to have many biological properties, including antiparasitic, antidiabetic, anticancer (Padhye et al., 2008) and diuretic (Zaoui et al., 2000) effects.Antibacterial and immunostimulant effects of BCS have also been reported (Khan et al., 2012).
Another plant of nutritional and medicinal importance is buckwheat (Fagopyrum esculentum Moench), an ancient dicotyledonous crop that belongs to the family Polygonaceae.Its medicinal properties are due the presence of bioactive compounds such as flavones, flavonoids, phytosterols, tocopherols, inositol phosphates, rutin and myo-inositol (Zhang et al., 2012).Additionally, buckwheat is rich in unsaturated fatty acids.Products made from buckwheat are efficient in lowering blood cholesterol concentrations, particularly of low density lipoproteins (LDL) and very low density lipoproteins (VLDL) (Tomotake et al., 2006), which are considered advantageous in preventing human disorders such a cardiovascular diseases, hypercholesterolemia and hypertension (Karamac, 2010).
In previous studies the authors observed that BCS-supplemented diets improved broilers' productive performance such as weight gain, feed intake, feed conversion ratio (FCR) and survivability, and at the same time suppressed harmful intestinal bacteria (Islam et al., 2011;Siddiqui et al., 2015).Similarly, the researchers found that BWS-supplemented diets in broiler feed significantly improved growth performance, increased serum high-density lipoproteins (HDL-cholesterol) and reduced serum total cholesterol, LDL cholesterol and triglyceride concentrations (Sayed et al., 2015).The antimicrobial effect of buckwheat has also been reported by Swiatecka et al. (2013).Although several reports have focused on the effects of supplementation of BCS or BWS in various animals, including poultry, none of the studies have evaluated the combined effect of BWS and BCS in broiler diets.Considering the medicinal advantages of BWS and BCS (above), the current study was designed to evaluate the combined effects of BWS and BCS on growth performance, serum biochemical metabolites and intestinal bacterial flora in broiler chickens.

Materials and Methods
One hundred and twenty day-old broiler chicks (Cobb-500) were purchased from a local hatchery.Initially, the chicks were reared in a brooding house for up to 10 days to adjust to environmental conditions.Then the birds were weighed and randomly assigned to five treatment groups of 24 chicks each.Each treatment consisted of three replicates of eight birds (four male and four female) each in a completely randomized design.Birds were provided ad libitum access to feed and water.Care and management of the birds adhered to the accepted guidelines (FASS, 2010).Chicks were vaccinated against Newcastle and Gambaro disease on the fourth and twentieth days, and were examined regularly for abnormal clinical signs (restlessness, lordosis, abnormal gait, vices and depression) as well as feed intake throughout the experiment.Throughout the study, the birds were housed according to standard temperature regimens, gradually decreasing from 32 ºC to 24 ºC.
A three-phase feeding programme was used, with a starter diet from days 1 to 10, a grower diet from days 11 to 20, and a finisher diet from days 21 to 40.All diets were formulated to meet or exceed the nutrient requirements of broilers, provided by National Research Council (NRC, 1994).Buckwheat seed (BWS) and black cumin seed (BCS) were obtained from a local market.The seeds were coarsely powdered with a mechanical grinder and then mixed directly with manually prepared diets.The birds were allocated to four treatments, and were fed a manually formulated control diet (no BWS and BCS) (T 1 ) or manually formulated diets supplemented with 10% BWS + 1.5% BCS (T 2 ), 20% BWS + 2.5% BCS (T 3 ) and 30% BWS + 3.5% (T 4 ).The experiment started on the tenth day and lasted a further 30 days.The composition of the grower and finisher diets used in the treatments is presented in Table 1.
Initial bodyweights of the birds were measured before starting the treatments.During the experimental period, growth performance was evaluated.Feed consumption and bodyweight gain were recorded regularly and FCR (feed intake/weight gain) was calculated.
Blood samples were collected from randomly selected birds of each replication at 15 days and at the end of the study (at day 30) by wing vein puncture with sterilized syringes and needles (Islam et al. 2011).These samples were centrifuged at 3000 rpm for 10 minutes to obtain serum.Individual serum samples were analysed for total cholesterol, high-density lipoprotein (HDL)-cholesterol and triglycerides concentrations, using the kit package manufactured by Crescent Diagnostics (K.S.A.).
Faecal samples of chickens were collected from healthy broilers during the last week of the experiment.Fifteen birds (one bird from each replication) were slaughtered, and faecal samples were collected from the large intestine of each bird with sterilized surgical instruments.Microbial populations were determined by serial dilution of faecal samples.Plate count agar, eosin methylene blue agar, salmonellashigella agar and Lactobacillus MRS agar media were used to determine total viable bacteria, E. coli, Salmonella sp. and Lactobacillus sp., respectively.The inoculated media were marked and incubated at 37 ºC for 24 -48 h.The number of colonies (30 -300) in a particular dilution was multiplied by the dilution factor and expressed as mean log 10 ± SD CFU/g.Colony characteristics such as shape, size, surface texture, edge and elevation and colour developed onto selective media after 24 -48 h of incubation at 37 ºC were recorded as described by Marchant & Packer (1967).All data were analysed with the SPSS statistical analysis programme (version 17.0).A P-value of <0.05 was considered to indicate a significant difference between groups, and a comparison of means was made using Duncan's multiple range test (Steel & Torrie, 1984).The results are expressed as average ± standard deviation of three replications (n = 3).Data points bearing different letters are significantly different (P <0.05).

Results
The effect of different dietary treatments on bodyweight gain, total feed intake and FCR of broilers are shown in Table 2. Bodyweight gain, total feed intake and FCR in various treatment groups were statistically significant (P <0.05).Bodyweight gain of broilers receiving 10% BWS + 1.5% BCS was statistically higher compared with the other treatments.Total feed intake and FCR of chicks receiving diet with BWS and BCS increased too.The effects of the experimental diet containing BWS and BCS on serum total cholesterol, HDLcholesterol and triglycerides concentrations of broilers at the 15th and 30th days of treatment are presented in Figure 1.The results showed that BWS diet supplemented with BCS significantly improved (P <0.05) these biochemical parameters.
Serum total cholesterol concentration of chicks fed BWS and BCS decreased (P <0.05) compared with the control groups at the 15th and 30th days of treatment (Figure 1-A).After 15 days, there was no significant difference between the levels of BWS and BCS supplementation, but at the end (30th day) they differed significantly (P <0.05).The lowest value (60.33 mg/dL) was recorded in birds fed 20% BWS supplemented with 2.5% BCS (T 3 ).HDL-cholesterol concentrations were significantly higher (P <0.05) in birds receiving diets with BWS and BCS (Figure 1-B).Values were almost non-significant up to 15 days and then increased significantly, irrespective of levels of BWS and BCS, than those of the control.Broilers receiving 10% BWS supplemented with 1.5% BCS (T 2 ) diet had the highest HDL-cholesterol concentration (42.7 mg/dL).
The reducing tendency (P <0.05) of serum triglycerides levels was also observed in the diets of BWS and BCS compared with the control (Figure 1-B).The lowest concentration of triglycerides was recorded (65.2 mg/dL) in 10% BWS supplemented with 1.5% BCS (T 2 ) diet.However, the differences observed among the levels of BWS and BCS did not show statistical significance.
Figure 2 shows the effects of varying levels of BWS and BCS-supplemented diets on the population of total viable bacteria, Escherichia coli, Salmonella sp. and Lactobacillus sp. in faeces of broilers.Buckwheat and supplementation of black cumin seed powder in the diets decreased (P <0.05) in the population of harmful bacteria, Escherichia coli, Salmonella sp. and total viable bacteria than in the control (no BWS and BCS).Total viable bacteria were found to be decreased from Log 10 11.73 CFU/g (T 0 ) to Log 10 10.90 CFU/g (T 4 ), E. coli from Log 10 9.18 CFU/g (T 1 ) to Log 10 9.09 CFU/g (T 4 ) and Salmonella sp. from Log 10 9.17 CFU/g (T 1 ) to Log 10 9.07 CFU/g (T 4 ).Although the population of beneficial bacterium, Lactobacillus sp. was increased in the BWS and BCS groups, these increments were not significantly different from the control.

Discussion
In previous studies, the authors observed that supplementation of BWS and BCS powder in diets significantly decreased serum total cholesterol, triglycerides and LDL-cholesterol concentrations, and the population of E. coli in broilers and layers (Islam et al., 2011;Sayed et al., 2015;Siddiqui et al., 2015).The findings of our current research are more or less in accordance to the findings of some earlier researchers (Guler et al., 2006;Ziad et al., 2008;Majeed et al., 2010).The current findings and earlier results (Islam et al., 2011;Sayed et al., 2015;Siddiqui et al., 2015) suggest that herbal feed additives might be an effective alternative to synthetic antibiotics for the promotion of health and performance of poultry.
Several studies have observed that BCS has potential as an alternative to antibiotics and vaccination to improve immunity and to reduce mortality in poultry birds owing to the presence of pharmacologically active constituents such as thymoquinone, dithymoquinone, thymohydroquinone, nigellicine, nigellimine and nigellidine (Abdur-Rehman & Abu-Bakar, 1997; Osman & El-Barody, 1999).BCS supplementation in broiler diets has been reported to strengthen the immune system by preventing lipid peroxidation and liver damage (Sogut et al., 2008), Rutin is the most influential pharmacological compound in buckwheat, and is reported to improve immunity (Sayed et al., 2015).The poor performance of chickens fed higher levels of buckwheat was probably due to the characteristics of hardness and springiness of the buckwheat flour and a high crude fibre content.Birds have limited ability to digest crude fibre owing to an insufficient amount of the cellulase enzyme in their digestive systems (Gupta et al., 2002).
According to the results on the lipid profile of broilers in this study it could be deduced that BWS and BCS have favourable effects on serum metabolites.Both BCS and BWS have been reported to possess hypolipidemic and hypocholesteremic properties in animal studies (El-Bagir et al., 2006;Sayed et al., 2015).
In the current study it was observed that BWS and BCS supplementation in broiler diets significantly decreased serum total cholesterol and triglycerides levels, but increased HDL concentrations compared with the control treatment (Figure 1).Similar effects of BCS and BWS on serum lipid profile had been obtained by earlier investigators (Tomotake et al., 2006;Islam et al., 2011;Sayed et al., 2015;Siddiqui et al., 2015;Siddiqui & Sayed, 2015).Al-Beitawi & El-Ghousein (2008) reported that feeding broiler chicks with BCS supplemented diets reduce plasma cholesterol and triglycerides compared with broiler chicks fed control diet.Reduced serum triglycerides and total cholesterol levels were observed, while the serum HDL cholesterol level increased owing to supplementation of layer rations with black cumin seeds (Akhtar et al., 2003;Siddiqui & Sayed, 2015).Furthermore, Sayed et al. (2015) observed similar results with BWS supplementation with chitosan in broiler diets.
Although the mechanism of lipid profile improvement is not clearly understood from the current study, this may be because of possible cholesterol-lowering mechanisms of tocopherols, such as inhibition of cholesterol oxidation (Xu et al. 2001) and reduced HMG-CoA-reductase activity (Ha et al. 2005).Furthermore, Martin et al. (2001) and Torra et al. (2001) hypothesized that the cholesterol-lowering mechanism of black cumin fixed oil is dependent on peroxisome proliferator-activated receptor (PPARα) activation.The volatile oils of black cumin contain quinines, including thymoquinone (TQ) and dithymoquinone, which might be involved in a sharp decrease in serum triglyceride level in hens fed a BCSsupplemented diet (Swamy & Tan, 2000).A serum triglyceride lowering effect of TQ has been reported in rats (Bamosa et al., 2009).In addition, the choleretic activity of the seed powder (El-Dhakhny et al., 2000), high level of polyunsaturated fatty acids (Ramadan, 2007) and β-sitosterol in black cumin oil make it effective in lowering blood cholesterol concentration and preventing coronary heart disease (Cheikh-Rouhou et al., 2008).On the other hand, the cholesterol-lowering effect of buckwheat is linked with lower digestibility of buckwheat and the presence of fibre-like substances, which is indicated by an increase in the levels of neutral and acid sterols in rat faeces observed when a diet rich in buckwheat protein products (Tomotake et al., 2001) is administered.
In the present study, a significant reduction (P <0.05) was observed in the population of harmful bacteria, E. coli and Salmonella spp., as well as total viable bacteria, in the intestine of the chicks fed BCSand BWS-supplemented diets compared with the control (without BCS and BWS).However, the population of beneficial bacterium, Lactobacillus sp., was not significantly affected by BWS and BCS supplementation.These findings are in agreement with those of Alsawaf & Alnaemi (2011) and Islam et al. (2011), who reported that black seed and oil inhibited the growth of E. coli, S. aureus, Brucella melitensis and total viable bacteria.Moreover, several studies have referred to the antibacterial effect of BCS and BWS against E. coli, Enterobacteria, Enterococcus, Streptococcus, Lactobacilus and Bifidobacteria (Cabarkapa et al., 2008;Halawani, 2009;Préstamo et al., 2014).Antimicrobial activity of BCS could be attributed to the presence of thymoquinone (TQ) and thymohydroquinone (THQ) in seed oil (Halawani, 2009) while flavonoids and polyphenols influence mainly the inhibitory effect of buckwheat on microorganisms.

Conclusion
In terms of the results obtained from the present investigation it could be stated that BWS-and BCSsupplemented diets improved performance, significantly decreased the levels of serum total cholesterol and triglycerides in broilers with an elevation of HDL-cholesterol and significantly suppressed the growth of pathogenic bacteria (E. coli and Salmonella spp.).Taken together, these results suggest that poultry diet supplemented with 10% buckwheat seed + 1.5% black cumin seed (T 2 ) could be regarded as natural feed additives and environment safe diets as alternatives to banned and hazardous synthetic antibiotics.However, further in-depth research is needed to assess the obscure mechanisms of serum cholesterol lowering and antibacterial functions of buckwheat and black cumin seed before they can be used as poultry feed additive.

Table 1
Ingredients and chemical composition of the grower diets fed to experimental broilers

Table 2
Growth performances of broiler chickens fed various experimental diets for 30 days a BWS: buckwheat seed; BCS: black cumin seed.*Body weight gain (g): final body weight at 40th day (g) -initial body weight at 10th day (g).**FCR: feed conversion ratio (total feed intake (g)/body weight gain (g)).