Effects of Nigella sativa on Performance, Blood Profiles, and Antibody Titer against Newcastle Disease in Broilers

Recent anxiety about resistance to chemical drugs has elevated the position of phytogenic feed additives including Nigella sativa in preventive strategy in the poultry industry. During this study, a completely randomized experiment was designed to investigate the efficacy of different levels (0 to 16%) of N. sativa seeds supplemented in the diet of broilers on performance, immune responses, and hematological and biochemical parameters. The results indicated the following: (a) Supplementation of 1% N. sativa seeds in diet had the highest positive effects and 16% N. sativa had the highest significant (p=0.03) adverse effects on weight gain, while up to 2% N. sativa seeds in the diet reduced feed conversion ratio (FCR) whereas 4% and over that increased the FCR. (b) Chickens fed with a diet containing 1% N. sativa seeds had the highest antibody titers, but those fed with 16% N. sativa seeds had the lowest antibody titers at end of the experiment. (c) Dietary inclusion of N. sativa seeds increased hemogram parameters and the group fed with 16% N. sativa seeds had the highest values on day 21 until the end of the experiment. (d) Supplementation of N. sativa seeds decreased in WBC and lymphocytes but increased heterophils, H/L, monocytes, eosinophils, and basophils percentages. Supplementation of up to 2% of N. sativa seeds in broiler's diets elaborated serum level of those parameters, while supplementation of ≥ 4% N. sativa seeds decreased their serum levels. In conclusion, supplementation of N. sativa seed (1-2%) in broiler diets, as a multipurpose natural growth promoter, improves performance, elevates humoral immune responses, affects serum biochemical profiles of broiler chickens, and induces changes in their hemogram and leukogram, while there are no side, residual, and hazardous effects.


Introduction
Phytogenic feed additives "phytobiotic" are assuming a position of prime importance in the poultry industry [1][2][3] and could be used in feed or water [4]. Among the promising phytogenic immune-stimulants, Nigella sativa (N. sativa) is a miracle medicinal herb having a long history with a rich religious background [5,6]. N. sativa seeds are used for the treatment of some disorders and illnesses including fever, common cold, headache, asthma, various Gram-positive and Gram-negative microbial infections [7], aflatoxicosis [8], antiethanol hepatotoxicity [9], and alleviation of adverse effects of heat stress [10], as well as to expel worms from the intestines. e possible biochemical active components of N. sativa and their pharmacological effects have been investigated [11,12], and their pharmacological actions as immune stimulation, antioxidant, anti-inflammatory, antimicrobial, and antiparasitic activities [5,[13][14][15][16][17] have also been studied. supplementation of N. sativa seeds to poultry diets could be recommended as a natural growth promoter instead of antibiotics. Previous reports indicate that diets containing different levels of N. sativa seeds improve the performance and carcass characteristics in broilers [5,21]. e inclusion of N. sativa seeds in layers' diet also increases egg production as well as eggs' quality [22] and serum triglycerides in layers [23]. However, N. sativa also affects blood profile and biochemical parameters [24,25] and prevents atherogenesis by decreasing LDL-cholesterol and increasing HDL cholesterol [26]. N. sativa can control infectious diseases and regulate adaptive immunity via enhancement of macrophages [27]. Effects of N. sativa on cell-mediated immunity [28] and humoral immunity [29] have also been investigated. Although it has been documented that N. sativa has immunomodulatory effects [5], the type of immune responses against various pathogens provoked by N. sativa may need further investigations. However, interest in herbal medicines and their multiple beneficial utilization in poultry health and performance has been recently reviewed [5,6,[30][31][32]. Finding the best beneficial and safe phytogenic feed additives as the most suitable substitute to antibiotic promoters is much more today's research topic [1,2,4,[33][34][35]. e objective of this research was a comprehensive study on the efficacy of N. sativa as a multipurpose feed additive on performance, blood profiles, and humoral immune responses in broiler chickens. To the best of our knowledge, the closest reports to our study in the literature are works of Al-Beitawi et al. [29], Durrani et al. [36], Shewita and Taha [37], Ghasemi et al. [38], Hossain et al. [39], Shirzadegan et al. [40], Singh and Kumar [41], and Aydogan et al. [42], and the present study has a lot of differences with them including the use of various percentages of N. sativa seeds in diets and weekly measurement of performance, immune responses, serum biochemical parameters, hemogram, and leukogram indices.

Ethics of Experimentation.
All experimental procedures were carried out according to the standard animal experimentation protocols of the Veterinary Ethics Committee of Faculty of Veterinary Medicine, Urmia University (Ref. IR-UU-AEC-3/902/DA).

Vaccines and Vaccination
Routes. According to breakthrough level 1:8 (log 2 -3 ) of maternal antibody [43], live ND clone 30 vaccine (eye-drop route) together with ND oil (subcutaneous route) is used for primary vaccination on day 9 of age, and the chickens were revaccinated with live ND clone-30 vaccine by eye-drop method [44] on the 21st day.

Sampling
Procedures. Blood samples for hematological tests (using EDTA anticoagulant-treated syringes) and blood samples for evaluation of immunity and biochemical tests were collected on day one and then at weekly intervals until the end of the experimental period (42 days old), as previously described [45]. Blood samples were labeled according to the number and date of sampling and kept at room temperature until clotted, and then, the sera were separated for serological tests.

Evaluation of Performance.
e performance of the chickens for each week was determined.

Evaluation of Antibody
Titer. On day one and day seven of age, serum samples were used to evaluate maternally derived antibodies of the chicks to determine the best time of first vaccination. On day 14 and weekly interval, serum samples were used to assess protective immune response derived from vaccination against ND by application of hemagglutination inhibition (HI) test as it has been reported that the HI test is an excellent indicator of the immune status and disease resistance of a flock, especially to assess protective responses following vaccination because, unlike ELISA, HI test correlates well with the more laborious virus neutralization (VN) assays [44].

Statistical
Analysis. e results were analyzed by using the SPSS software (Version 21; SPSS Inc., Chicago, USA). e means for the treatments showing significant differences in the ANOVA were compared using the Post Hoc Tukey and Wilcoxon (nonparametric tests, 2 related samples) tests used for analyzing data of each parameter within the group and between groups. e relationship between age and parameter contents was ascertained through the Spearman correlation coefficient test. Differences were considered significant at p < 0.05.

Results
3.1. Performance. N. sativa had some effects on the performance of the chickens (Table 2), and as shown in Figure 1, the chickens of group T3 fed with a diet containing 1% N. sativa seeds had the highest weight at the end of the experiment but differences with those of the groups (T0-T5) were not significant (p > 0.05), but those with those of the groups (T6 and T7) were significant (p < 0.05). Dietary inclusion of 16% N. sativa seeds had a significant (p < 0.03) depressive effect on weight gain of chickens (T7) during the experimental period. As shown in Table 2, the feed efficiency ratio was affected by the level of N. sativa seeds, and supplementation (T3 and T4) of the diet with 1%-2% N. sativa seeds had decreased FCR but 8%-16% increased FCR. Figure 2, maternal antibody titer (MDA) of the chickens against ND decreased gradually and reached the points that are considered as negative in the control group T0 (environmental contamination control) on day 21 of age, and the comparison of average antibody titers of the groups during 2 weeks of age could be explained where maternal antibody of the chick's yolk may contribute to MDA of chicks during 3 days of age, and reduction of the MDA starts on day 4 of age based on half-life time (4-5 days) in case of ND [42]. On the contrary, antibody titers of ND of the vaccinated chickens (group T1-T7) were increased after vaccination at the 14th day of the experiment. Different levels of N. sativa seeds in diet affected antibody titers against ND in the vaccinated groups and the chickens (group T3) fed with 1% N. sativa supplementation had the highest level of antibody titers, while chickens of group T7 (16% N. sativa seed in diet) had the lowest level of antibody titers on days 28, 35, and 42 of age ( Figure 2). Weekly comparison of antibody titers of the groups revealed the following: (1) Differences among the groups were not significant (p < 0.05) on days 1, 7, and 14 of  vaccinated groups at the end (day 42) of the experiment showed that antibody titers of group T3 (vaccinated and feed with a diet containing 1% N. sativa seeds) differed significantly (p � 0.027) from that of group T1 (vaccinated but feed without N. sativa seeds diet) and from those of group T6 (vaccinated and feed with diet containing 8% N. sativa seeds) and group T7 (vaccinated and feed with the diet containing 16% N. sativa seeds) significantly (p � 0.02 and p � 0.01, respectively) indicating effectiveness of different levels of N. sativa seeds supplementation in diets, as dietary inclusion of up to 1% N. sativa seeds had the best immunomodulatory effects on the level of antibody titer following vaccination. Table 3, RBC and PCV as well as Hb values are age-dependent, and their values increased as the chickens get older. Weekly comparison of these parameters showed that supplementation of N. sativa seeds in the diet increases the values of RBC, PCV, and Hb, and the group of chickens fed with 16% supplemented N. sativa seeds had the highest values and significantly (p < 0.05) differed from those of control groups (T0 and T1) as well as those of the treated groups (T2-T4) indicating that increasing effects of N. sativa seeds on hemogram parameters are dose-dependent (Table 3). Table 4, WBC count of the vaccinated control 2 group (T1 � V + N−) was higher (p > 0.05) than that of the control 1 group (T0 � V−N−) from day 14 up to the end of the experiment indicating that WBC counts were affected by ND vaccination. Supplementation of N. sativa seeds affects leukogram parameters by decreasing WBC counts and lymphocytes and increasing heterophils, H/L ratio, monocytes, eosinophils, and basophils percentages from day 21 of age until the end of the experiment. Weekly comparison (Table 4) of the groups indicates that the group (T7) fed with 16% N. sativa seeds had the lowest WBC counts and lymphocyte percentages and their differences with control 2 group (T1 � T0 + V) were significant (p � 0.013 and p � 0.008, respectively). But, values for H/L ratio and percentages of heterophils, monocytes, eosinophils, and basophils of the group fed (T7) with 16% N. sativa seeds were the highest and their values differed from those of the control 2 group (T1 � T0 + V) significantly (p � 0.001).

Albumin and Total
Protein. e concentration of albumin and total protein of control chickens increased during 6 weeks of the experimental period indicating that    Evidence-Based Complementary and Alternative Medicine these parameters are age-dependent. A weekly comparison of the groups (Table 5) showed that supplementation of up to 2% of N. sativa seeds in the diet increases serum level of these parameters whereas 4% and over that decrease concentrations of these elements in the serum of broiler chickens. Overall, albumin and total protein levels of chickens fed with a diet containing different N. sativa seeds were higher than those of the control groups (T0-T1) and the differences were significant (p < 0.05) when compared with those of the control groups (T0-T1). Table 5, weekly comparison of the control groups (T0-T1) revealed that the serum glucose concentration of the chickens is decreased by age, and vaccination against ND did not affect its level. A weekly comparison of the treated groups indicated that the effects of N. sativa on serum glucose were dose-dependent. On the other hand, increasing the effect on serum glucose of the chickens was observed by 0.5 to 2% of N. sativa seed supplementation in diet, while serum glucose of the chickens fed with 4% and over that decreased gradually (Table 5). Overall, the chickens fed with different concentrations of N. sativa seeds in the feed had higher glucose level than those of the control groups (T0-T1), and a weekly comparison of the serum glucose level of the groups showed that on age 14 up to the end of the experiment, chickens of the group (T4) fed with 2% N. sativa diet had the highest level of serum glucose and differences between the groups were significant (p < 0.05). Table 5, supplementation of up to 2% of N. sativa seeds in diet had increasing effects on levels of serum calcium and phosphorus, while 4% and over that had decreasing effects. Chickens fed with 2% of N. sativa seeds in the diet had the highest level of serum calcium and phosphorus that significantly (p < 0.05) differed from those of the control groups (T0 and T1). Table 6, high levels of cholesterol and triglyceride due to yolk residue reduced sharply in the 1 st week, and then their levels reduced gradually during the experimental period, indicating that their concentrations are age-dependent at least in broilers up to 42 days of age. Weekly comparison of N. sativa treated and control groups showed that inclusion of N. sativa seeds in broiler's diets reduces the concentration of both these parameters, and chickens of group T7 fed with 16% N. sativa seeds had the lowest level and differed significantly (p < 0.05) from those of the control groups (T0-T1). Table 6, the lack of significant differences between group T0 and group T1 indicates that vaccination does not affect the serum enzyme levels, and levels of both ALP and AST were decreased by age, while those of ALT were increased. Weekly comparison (Table 6) of the groups revealed the following. (a) e levels of ALP decreased by the inclusion of N. sativa  Evidence-Based Complementary and Alternative Medicine groups revealed that the decreasing effect of N. sativa on the AST level is dose-dependent and group T7 had the lowest levels during the whole experimental period. As shown in Table 6, the mean differences of the groups T6 (8%) and T7 (16%) significantly (p < 0.05) differed from those of the rest groups on day 21of age to the end of the experimental period.

Performance.
Comparison of the vaccinated group (T1) with the unvaccinated group (T0) indicated that vaccination had a little (p > 0.05) adverse effect on weight gains of the chickens as expected due to vaccination stress. As shown in  Figure 1, our results about the efficacy of N. sativa supplementation on the performance of broiler chickens are in agreement with the results obtained by Miraghaee et al. [24], Ghasemi et al. [38], Hossain et al. [39], and Ali et al. [47] who reported that 1% N. sativa seeds in diet improved the performance of broiler chickens but there are some differences between our results with those obtained by Shewita and Taha [37] who observed a higher weight in chickens fed with 2% N. sativa seed supplementation diet when compared with the weight of those that received 10% of N. sativa seeds in their diet, or Durrani et al. [36] who reported a higher weight gain in 40 g/kg (4%) of N. sativa seeds in the diet.
Overall, most studies indicate that a higher percentage of N. sativa seeds in the diet may reduce feed intake and finally affects the average weight of broiler chickens as observed in this study (Table 2) for groups T6 (8% N. sativa seeds) and T7 (16% N. sativa seeds), and the mean of the latest differed significantly (p < 0.05) from those of the control (T0-T1) groups ( Figure 1).

Immune
Response. e lack of an increase in antibody titer of chickens of the unvaccinated group (T0) during the experimental period confirmed that no environmental or cross-contamination has occurred. Lack of significant differences among treated groups during days 1-14 of age indicates that supplementation of N. sativa seeds on diet had no significant effects on MDA reduction of the chickens. A significant (p � 0.04) difference between antibody titers of group T3 and group T1 indicates that dietary inclusion of 1% N. sativa seeds had the highest immunomodulatory effects, while that of group T7 (16% N. sativa seeds) had immunosuppressive effects. e results observed during this study ( Figure 2) indicate that N. sativa is able to promote antibody response (mostly IgG) in chickens as previously reported [5,17,29,36,47,48]. Higher antibody titers (log 2 -7.4 ) of chickens (group T3) observed during this study are in the range of expected titers that could be induced via vaccinations by two live plus one killed vaccines [49,50]. Our results about the efficacy of different levels of N. sativa seeds in the diet on immune responses of chickens also is in harmony with the results obtained by Hossain et al. [39] who reported that supplementation of 1% N. sativa seeds in a broiler diet improved the development of immunity but is in disagreement with the results obtained by Khan et al. [51]. However, our observation on antibody titers is in disagreement with those of Shewita and Taha [37] and Ghasemi et al. [38] who reported that 20 g/Kg of N. sativa had the highest immunomodulatory effects, and this inconsistency could be attributed to the lack of 1% concentration in their work. Our observation on a decrease in lymphocytes percentage together with an increase in antibody titers following vaccination against Newcastle diseases is controversial to the results obtained by Islam et al. [52] who reported a decrease in antibody titer together with a rise in peripheral lymphocytes with an explanation that the numbers of antigen-specificantibody secreting B-cells were less. As recent reports indicated that N. sativa has induced a significant increase in antibody titers against Newcastle disease (ND) and infectious bursal disease (IBD) in broilers [29,36], therefore, the reasons for this controversy results may be due to the nature of pathogenic agents, type of treatments (vaccination versus challenge), kind of experimental animal, different components and dosage of N. sativa, and the role of macrophages in the activation of 2 cells and their roles in the activation of B-cells for further differentiation into plasma cells. For example, the increase in antibody titer against avian infectious bronchitis (IB) has been observed by Durrani et al. [36] but has not been observed by Al-Beitawi et al. [29] or various dosages of N. sativa had different effects on antibody titers against different pathogenic agents (ND, IBD, and IB) in same experimental animals "broiler chickens" [36]. Table 3, the lack of significant (p > 0.05) differences between hemogram values of the control 1 (T0) and those of control 2 (T1) groups indicates that vaccination alone did not significantly influence the hemograms. e results (Table 3) obtained during this study in regard to values of some hematological parameters (RBC, PCV, and Hb) in broiler chickens are in agreement with those previously reported [53]. (Table 4) on the reduction effects of N. sativa on leukocyte counts of the chickens are in agreement with previous reports [54,55]. e results obtained during this study revealed that supplementation of N. sativa seeds in broiler's diet increased the H/L ratio (Table 4), and our results are in disagreement with the results obtained by Ali et al. [26] who reported that supplementation of 1% N. sativa may reduce the heterophil/lymphocyte (H/L) ratio. An increase in eosinophil values observed during this study (Table 4) is in line with the results obtained by Ahmad et al. [13] who reported that N. sativa rises peripheral blood lymphocytes and eosinophils in Long-Evans rats. It has been documented that monocytes-macrophages play an indispensable role in the immune system [55] and an increase in their numbers may indicate the immunomodulatory effects of N. sativa.

Biochemical Parameters.
In general, biochemical parameters of poultry may be influenced by various factors and the results of this study on biochemical parameters (Table 5) were compared with reported reference ranges [40][41][42][56][57][58]. Table 5, alternation on concentrations of albumin and total protein of the chickens fed with N. sativa seeds observed during this study have also been reported by Miraghaee et al. [24]. However, weekly comparison (Table 5) values of total protein and albumin of the treated groups showed that differences among the groups were not significant (p > 0.05) as previously reported [42,59] but in disagreement with the results observed by Singh and Kumar [41]. As shown in Table 5, increasing of protein and albumin concentrations of the chickens fed with 0.5-2% and decreasing of those chickens fed with 4-16% N. sativa may indicate that the Evidence-Based Complementary and Alternative Medicine efficacy of N. sativa on protein and albumin concentrations is dose-dependent.

Glucose.
e results obtained during this study (Table 5) on the level of serum glucose of chickens are in agreement with recent reports [29,40] but in disagreement with other reports [41,42]. Some controversial observations could be explained where the type of the extract, whole seed, and duration of consumption may exert different effects on glucose level as has been observed by Mohtashami [60] who reported that N. sativa did not significantly affect the fasting blood glucose level but significantly decreased its levels during oral glucose tolerance test (OGTT) when compared to diabetic control by affecting the time course of glucose absorption from the intestine.

Calcium and Phosphorus.
e results obtained for calcium and phosphorus during this study (Table 5) are in agreement with previous reports that supplementation of N. sativa in the feed of broiler diets significantly increases the level of serum calcium [61,62] because N. sativa possesses estrogenic activity in regulating calcium-regulating hormones [61].

Cholesterol and Triglyceride.
Because of yolk sac in day-old chicks, the concentration of both cholesterol and triglyceride were very high at the beginning of the experiment, but as shown in Table 6, by aging, their values reduced, and their levels in the control groups at age 42 days were in the range as previously reported [63]. e results obtained during this study on the serum cholesterol and triglyceride levels of broiler chickens are in agreement with previous reports that N. sativa reduces serum cholesterols of chickens [21,24,29,[37][38][39][40]47], in particular HDL cholesterol [26]. e reduction in serum cholesterol and triglycerides has been attributed to the lowering effect of black cumin (thymoquinone and monounsaturated fatty acids) on the synthesis of cholesterol by hepatocytes or fractional reabsorption from the small intestine [29]. It is also believed that phytosterols of N. sativa inhibit cholesterol absorption and may interfere with the reabsorption of endogenous cholesterol [26]. Moreover, it has also been reported that N. sativa decreases plasma concentrations of cholesterol by stimulating bile acid excretion [5], and a reduction in plasma cholesterol level could also be attributed to the effects of black cumin on the cholesterol excretion into the intestine [21,26]. As shown in Table 6, the lowering effects of N. sativa supplementation in the diet of commercial layers on cholesterol could be useful because lower egg yolk cholesterol of commercial layers' eggs is highly desirable for human consumption [5], but it may not be useful in breeders because of egg yolk role in embryo development.

Serum
Enzymes. Serum enzymes including ALP, ALT, and AST are mainly monitored for the evaluation of liver damage [3,9], and antioxidant activity of N. sativa components such as thymoquinone may play a vital role in the prevention of liver tissue [64]. e reduction of ALP and AST levels of broiler chickens fed with N. sativa supplementation observed during this study (Table 6) is in agreement with previous reports [21,40,59] but is in disagreement with that observed by Shewita and Taha [37] who reported that serum glutamic pyruvic transaminase (SGPT) level significantly increased with N. sativa supplementation. As shown in Table 6, increasing effects of N. sativa seeds on levels of ALT observed during this study have also been reported [40] and may indicate positive effects of N. sativa on liver health because ALT level is commonly used clinically as a biomarker for liver health.

Conclusion
Supplementation of N. sativa seed (1-2%) in broiler diets, as a multipurpose natural growth promoter, improves performance, elevates humoral immune responses, affects serum biochemical profiles of broiler chickens, and induces changes in their hemogram (Hb, PCV) and leukogram, while there are no side, residual, and hazardous effects.

N:
Nigella V: Vaccinated RBC: Red blood cells PCV: Packed cell volume MCV: Mean corpuscular volume MCH: Mean corpuscular hemoglobin MCHC: Mean corpuscular hemoglobin concentration WBC: White blood cells.

Data Availability
Data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding this mansucript.