ABSTRACT
This study was performed to evaluate the effects of hydroalcoholic extracts of Withania somnifera (WS) root, Withania coagulans (WC) fruit, and 1,25-dihydroxycholecalciferol [1,25(OH)2D3] on immune response and small intestinal morphology of broiler chickens. A total of 550 male one-day-old Ross 308 broiler chickens were randomly allotted to 55 pens, with 10 birds per pen (replicate), and reared for 42 days. Broiler chickens were fed 11 dietary treatments including a positive control diet with adequate Ca; a negative control diet (Ca concentration reduced by 30%); and the negative control diet supplemented with either WS or WC extracts at three levels (0, 75, and 150 mg/kg diet) or 1,25(OH)2D3 at two different levels (0 and 0.5 μg/kg diet). Antibody response against sheep red blood cells (SRBC) was measured on days 32 and 39. Administration of 150 mg/kg WS and WC significantly increased the production of IgG specific to SRBC on primary and secondary antibody response, respectively. Birds given a negative control diet supplemented with only 150 mg/kg WS displayed the highest villus width compared to those given 75 mg/kg WS or WC with 1,25(OH)2D3. The present study showed that supplementation of WS or WC at 150 mg/kg had beneficial effects on humoral immune response. Also, administration of WS significantly increased villus width.
1. Introduction
Vitamin D3, also known as cholecalciferol, is generated in the skin of animals and is the inactive form of vitamin D. The active form of vitamin D is produced in the body in a two-step process. This process involves hydroxylation of the vitamin D3 molecule at the position of 25 in the liver to produce 25-OH-cholecalciferol (25-OH D3), the major circulating form of vitamin D3 in the blood. The circulating 25-OH D3 is then transported to the kidneys and hydroxylated at position 1 to produce 1,25-dihydroxycholecalciferol [1,25(OH)2D3], the most biologically active, hormonal metabolite of the vitamin D3 (Norman Citation2008). Vitamin D and its metabolites have receptors in the cells of the immune system such as monocytes and macrophages in addition to intestine, bone, and kidney (Aslam et al. Citation1998). Epidemiological studies in mammals demonstrated the role of vitamin D in modulating the immune response (Peelen et al. Citation2011). Previous studies with broiler chickens indicated that deficiency of vitamin D decreased cell-mediated immunity, including thymus weights, cutaneous basophil hypersensitivity (CBH) response, and phagocytosis by sephadex-elicited abdominal macrophages, with no effect on humoral immunity (Aslam et al. Citation1998). 1,25(OH)2D3 increases the development and secretion of Th2 cells, which induce B-cell growth and differentiation, and subsequently stimulate immunoglobulin production (Beal et al. Citation2004). The effects of Vitamin D on regulating the morphological and functional development of intestinal villus mucosa have been well documented (Shinki et al. Citation1991). Tabor and Tabor (Citation1984) reported that polyamines (putrescine, spermidine, and spermine) play an essential role in cell proliferation and differentiation. It has been shown that a single injection of 1,25(OH)2D3 into chickens deficient in vitamin D produced a marked increase in putrescine accumulation from ornithine and spermidine in the duodenum (Shinki et al. Citation1985). Calcium ions have an essential biological role in activation and maturation of lymphocytes (Berridge et al. Citation2003; Feske Citation2007). It has been shown that T and B lymphocytes have essential functions in cellular and humoral immunity, respectively (Silberman et al. Citation2002). In addition, Ca2+ ions are essential for production of interleukin-2 (IL-2) by T cells (Negulescu et al. Citation1994). Much attention in recent years has been focused on extracts of plant origin, mainly for their actions as antioxidant agents. There are 23 known species of Withania (Solanaceae plants) and among those only 2, Withania somnifera (WS) and Withania coagulans (WC), are economically important and widely cultivated (Panwar & Tarafdar Citation2006). Withania somnifera L., (WS) (Solanaceae) is an annual herb and a rich source of bioactive compounds. Several pharmacological activities of the plant have been attributed to its roots (Khan et al. Citation2009). Withania coagulans L. Dunal is a small evergreen shrub that has been shown to possess varied medicinal properties as a remedy for dyspepsia, flatulent colic, and other intestinal diseases (AbouZid et al. Citation2010). Previous chemical investigation of this plant resulted in identification of several withanolides from WC fruits (Atta-ur-Rahman et al. Citation2003; Prasad et al. Citation2010). Medicinal properties of both plants mentioned above are attributed to a group of steroidal lactones called withanolides, comprising a group of C-22 and C-26 patterns (Dewir et al. Citation2010). It is reported that withaferin A and withanolid E induced B and T cells’ proliferation, promoted selective enhancement of Th1 cell-mediated immunity, macrophages activation, and immunoglobulin response (Shohat et al. Citation1978). It has been shown that medicinal plants which are effective in growth promotion and immunostimulation have also beneficial effects on villi and epithelial cells’ structure and function (Yamauchi et al. Citation2006). In an experiment, Davis and Kuttan (Citation1998) found that administration of root extract of WS to mice whose immune systems were suppressed by cyclophosphamide significantly restores the normal structure of intestinal villi. The objective of this study was to evaluate the effects of 1,25(OH)2D3, hydroalcoholic extracts of WS root and WC fruit on broilers’ immune response and small intestinal morphology when used as supplements in the negative control diets (Ca concentration reduced by 30%).
2. Materials and methods
2.1. Preparation of plant extracts
Dried fruits of WC were purchased from the local market and the roots of WS grown in natural habitat were collected during the month of October from Saravan, Sistan, and Baluchestan, Iran. The roots and fruits were correctly identified and authenticated at the Herbarium of Botany Directorate in University of Sistan and Baluchestan, Iran. The fruits were coarsely powdered and soaked in 50% ethanol with occasional shaking at room temperature. Roots were thoroughly washed with sterile water, air-dried, and ground into a fine powder form. After 3 days, the ethanol soluble materials were filtered and concentrated using a rotary evaporator (Laborota 4000, Heidolph Germany), and then freeze-dried for 24 h to yield extracts. Dried extracts were stored at −20°C until used for experimental work.
2.2. Birds, diets, and experimental design
A total of 550 male one-d-old Ross 308 broilers were obtained from a commercial hatchery and reared in 55-floor pens with wood shavings litter at a stocking rate of 10 birds per pen (1 × 1 m). Feed and water were provided ad libitum throughout the 6-week experimental period. The temperature and lighting regime were controlled according to Ross broiler breeder instructions (Ross Citation2007). A completely randomized design was used with 11 dietary treatments replicated in 5 pens each. The dietary treatments were as follows: basal diets (positive and negative control) and the negative control diet supplemented with either WS or WC extracts at three levels (0, 75, and 150 mg/kg diet) or 1,25(OH)2D3 at two different levels (0 and 0.5 μg/kg diet). The compound 1,25(OH)2D3 (Sigma Aldrich, St. Louis, MO, USA) was used in liquid form using corn oil as a carrier (10 μg/ml). Both control diets () were based on corn and soybean meal and were formulated to meet the requirements suggested by the Ross 308 broiler nutrient specifications (Ross Citation2007) for all nutrients except Ca, which was reduced by 30% (obtained by reducing limestone and adding fine sand) in the negative control diet. Feed intake (FI) and body weights were recorded at 1, 11, 24, and 42 days of age, and body weight gain (BWG) and feed conversion efficiency (FCE, kg feed per kg gain) were calculated. All the research procedures were approved by the Animal Use and Care Committee of the Ferdowsi University of Mashhad.
Table 1. Composition of the basal diets (g/kg).
2.3. Experimental procedures
2.3.1. Immune response
2.3.1.1. Humoral immunity
Sheep red blood cells were administered as a test antigen to quantify specific antibody responses. On day 25, two birds per replicate were bled by cervical venipuncture, and 3 mL of blood was collected from each bird for prechallenge antibody titre analysis. This procedure was followed to check the presence of antibodies before challenging with SRBC. The same birds were then immunized intravenously via the brachial vein with 1 mL of 7% suspension of SRBC in 0.85% saline. Seven days postinjection, all birds were bled by brachial venipuncture, 3 mL of blood was collected for primary antibody response, and the antigenic challenge was repeated. The blood was left at room temperature for 2 h to clot and placed in a 4°C refrigerator overnight for maximum sera yield. The serum samples were collected and kept frozen at −20°C until serological analysis. Blood samples were collected 14 days after the first challenge to determine secondary antibody response. Total antibody response, IgM, and IgG were determined using microhemagglutination assay in 96-well plates as previously described (Lepage et al. Citation1996). Briefly, 2-mercaptoethanol-resistant antibodies (presumably IgG) were determined by incubating 0.05 mL serum with an equal volume of PBS (pH 7.5) and 0.2M 2-mercaptoethanol at 37°C for 30 min prior to the hemagglutination test. The 2-mercaptoethanol-sensitive antibody (presumably IgM) levels were determined by subtracting the 2-mercaptoethanol-resistant antibody titres from the total titres. The antibody titres were expressed as the log 2 of the highest dilution of serum that agglutinated 0.05 mL of 2.5% suspension of SRBC in PBS.
2.3.1.2. Cell-mediated immunity
The cell-mediated response of the birds was evaluated using a CBH response according to a procedure described previously (Kean & Lamont Citation1994). On day 37, two birds per replicate were injected intradermally in the interdigital skin between the second and third digits of the right foot with 100 μg of phytohemagglutinin-P (PHA-P) in 100 μL of PBS. The left foot received 100 μL saline and served as the control. Thickness was measured at 12 and 24 h later using a digital micrometer with an accuracy of ±1 mm (series 500, Mitutoyo, Tokyo, Japan). The swelling response was measured by subtracting the preinjection thickness from the postinjection thickness.
2.3.2. Lymphoid organ weights
On days 21 and 42, one bird per replicate was killed. Thymus, spleen, and bursa of Fabricius were collected and weighed.
2.3.3. Intestinal morphology
On days 21 and 42, five birds per treatment (1 bird/replicate) were selected randomly and slaughtered by cervical dislocation. Segments of the intestine were removed from the jejunum (2 cm in length) rinsed with 0.01 M PBS (pH 7.2), and placed into 10% buffered neutral formaldehyde solution (pH 7.2–7.4). Then, all samples were gradually dehydrated, sectioned at 6 mm. The tissues were processed, embedded in paraffin, and subsequently cut into 5 μm-thick slices that were placed onto slides. The tissues were stained with hematoxylin-eosin and observed using an optical microscope (Olympus BX41TF, Tokyo, Japan) coupled to a digital camera (Olympus DP12 U-TV0.5 XC-2, Japan) and connected to an image analysis system (Olysia Soft Imaging System, Germany). The histological parameters analysed were as follows: villus height, villus width, crypt depth, muscle depth, and villus height:crypt depth ratio.
2.4. Statistical analysis
Data were analysed using the general linear model ANOVA (SAS Institute Citation2003) in a completely randomized design. Means were compared using Duncan’s multiple range test. All differences were considered significant at p ≤ .05.
3. Results
3.1. Growth performance
Dietary treatments did not significantly affect BWG, FI, and FCR of chickens during the experiment ().
Table 2. Effect of WS, WC, and 1,25(OH)2D3 in negative control diets on broiler BWG, FI, FCE (kg feed per kg gain) at 42 d of age.
3.2. Immune response
3.2.1. Humoral immunity
Titres of anti-SRBC blood cells in the experimental groups are presented in . Supplementation of WS or WC extracts at levels of 75 or 150 mg/kg and 1,25(OH)2D3 did not significantly affect primary or secondary antibody responses of IgM and Ig total against the SRBC. However, a significant difference was observed in IgG between groups at both primary and secondary antibody responses. The dietary supplementation of hydroalcoholic extract of WS before SRBC immunization had a positive effect on the IgG-mediated response because a significant increase in IgG levels in response to SRBC was observed in birds fed negative control diets supplemented with 150 mg/kg compared to those fed 75 mg/kg of WS extract without 1,25(OH)2D3. In the secondary antibody response, the highest values of IgG levels were noted for birds which received 150 mg/kg of WC extract or 1,25(OH)2D3 alone. Results of the current study showed that birds receiving the diet with 150 mg/kg of WS had significantly higher titres of IgG at earlier ages, but those receiving the diets with 150 mg/kg of WC had a significantly higher response for IgG antibody during the secondary challenge.
Table 3. Effect of WS, WC, and 1,25(OH)2D3 in negative control diets on primary and secondary antibody responses.
3.2.2. Cell-mediated immunity
Dietary treatments did not affect CBH responses to PHA-P ().
Table 4. Effect of WS, WC, and 1,25(OH)2D3 in negative control diets on swelling response to phytohematoglutinin.
3.3. Lymphoid organ weights
The relative weights of lymphoid organs for chickens in various groups are summarized in . Lymphoid organ weights were expressed as a percentage of body weight. None of these organs (thymus, spleen, or bursa) were significantly affected by the plant extracts and 1,25(OH)2D3 at 21 days of age. At 42 days of age, dietary treatments only affected the relative weight of spleen. There was a significant difference among the spleen weight of chickens in various groups. The spleen weight in the 150 mg/kg WS and 1,25(OH)2D3 supplemented birds was significantly higher than those fed negative control diet supplemented only with 1,25(OH)2D3. Also, birds that received a negative control diet supplemented with WC significantly showed lower spleen weight with the exception of those that received 150 mg/kg WC combined with 1,25(OH)2D3.
Table 5. Effect of WS, WC, and 1,25(OH)2D3 in negative control diets on relative weight of the thymus, spleen, and bursa of fabricius of broilers at 21 and 42 days of age.
3.4. Intestinal morphology
The effects of the different dietary treatments on small intestinal morphology at 21 and 42 days of age are presented in and . Experimental treatments did not affect small intestinal morphologic parameters at 21 days of age. At 42 days of age, dietary treatments only significantly affected the villus width. The lowest values of villus width were obtained in birds that received 75 mg/kg WS or WC with 0.5 μg/kg 1,25(OH)2D3 (100.76, and 102.03 μm respectively). Birds given negative control diet supplemented with only 150 mg/kg WS displayed the highest villus width (160.07 μm).
Table 6. Effect of WS, WC, and 1,25(OH)2D3 in negative control diets on the small intestinal morphology of broilers at 21 days of age.
Table 7. Effect of WS, WC, and 1,25(OH)2D3 in negative control diets on the small intestinal morphology of broilers at 42 days of age.
4. Discussion
In a study conducted on chickens, Mirakzehi et al. (Citation2013) showed that dietary supplementation of 1,25(OH)2D3 and hydroalcoholic extract of WS at the level of 0.5 μg/kg and 75 or 150 mg/kg, respectively, had no effects on BWG, FI, and FCE. In another study conducted on laying hens in agreement with the findings of current study it was reported that a diet supplemented with 65 or 130 mg/kg of WS extract had no significant effects on FI and FCE (Tahmasbi et al. Citation2012). In contrast to our results, Alba et al. (Citation2015) reported that supplementation of WC fruit powder into the diet at 2.5 and 5 g/kg affected FI and BWG. They pointed out that as dietary inclusion of WC increased from 0 to 5 g/kg, both FI and BWG increased. The present results are not consistent with those obtained by previous research. It seems that the effects might be attributable to different forms of WC fruit which are used in experiments. As presented in , supplementation of 150 mg/kg WS or WC and 1,25(OH)2D3 resulted in significant increase of IgG antibody response between groups at both primary and secondary antibody responses. These results are in agreement with other studies reporting the immunostimulatory actions of 1,25(OH)2D3, WS, and WC (Garlich et al. Citation1992; Davis & Kuttan Citation2000; Cantorna et al. Citation2004; Malik et al. Citation2007). It has been shown that the active form of vitamin D, 1,25(OH)2D3, inhibits the development of autoimmune diseases, including inflammatory bowel disease (IBD) (Cantorna et al. Citation2004). An additional factor that determines the effect of vitamin D status on immune function is the dietary calcium. Dietary calcium has independent effects on IBD severity. Vitamin D-deficient mice on low-calcium diets developed the most severe IBD, and 1,25(OH)2D3 treatment of mice on low-calcium diets improved IBD symptoms (Cantorna et al. Citation2004). These results are in agreement with the findings of Garlich et al. (Citation1992) who observed that turkeys fed diets supplemented with calcitriol in addition to vitamin D3 (4000 IU/kg) had greater antibody response compared to controls that received vitamin D only. In accordance with the results of the current experiment, increasing the circulating antibody titre and antibody forming cells by supplementing of WS extract was reported earlier by Davis and Kuttan (Citation2000). These results are in agreement with Malik et al. (Citation2007) who showed that IgG and IgM titres’ values were significantly increased in mice with 30 mg/kg body weight of aqueous alcoholic (1:1) root extract of WS orally for 15 days. Malik et al. (Citation2007) reported that hydroalcoholic extract of WS root exerts a direct effect on increase of T and B cell proliferation, supported selective Th1 type immunity, and enhanced macrophage activation and immunoglobulins secretion and thereby stimulates cell-mediated and humoral immunity. Our findings suggested that WS extract is able to potentiate the humoral response. It has been shown that withanolides particularly withaferin A and withanolide E which are naturally occurring in WC extract have potent antitumor, antibacterial, antifungal, antioxidant, anti-inflammatory, and immnomoudulatory activities (Gupta et al. Citation2003; Leyon & Kuttan Citation2004). It was found that coagulin-H which naturally exists in WC extract has an effective role in lymphocyte proliferation and Th1 cytokine production. These cytokines, in turn, help to control the proliferation and differentiation of cells in the immune response (Mesaik et al. Citation2006). A possible mechanism involved in the increased levels of IgG produced after antigenic stimulation in birds fed higher doses of WS and WC extracts is probably related to increased expression of IL-2 and interferon-γ because these cytokines stimulate antibody production in birds (Wang et al. Citation2006).
It is well documented that the CBH response elicited in chickens by an intradermal injection of PHA-P is a thymus-dependent response mediated by T cells (Corrier & DeLoach Citation1990). This observation implies that supplementation of plant extracts and 1,25(OH)2D3 did not affect T cell-mediated immunocompetence in chickens. Liljequist et al. (Citation1992) reported that the interdigital skin test was a valid test for CBH responses in 12- to 19-d-old broiler chickens. In the current experiment, this test was performed on 37-d-old chickens. The results may be due to differences in age or genetics of the experimental birds.
Spleen is one of the important lymphoid organs involved in the development and differentiation of T or B lymphocytes (Eerola et al. Citation1987). White et al. (Citation1975) reported that spleen is the major source of antibody production against any antigen and its weight gain is a sign of increasing of the production of immune cells and cell population. In agreement with the results of this experiment, Davis and Kuttan (Citation1998) reported that administration of WS extract (20 mg/kg body weight) significantly increased the size and weight of spleen in rats with chemically suppressed immunity. Davis and Kuttan (Citation2000) reported that administration of 20 mg/kg body weight of WS root powder stimulated the weight of spleen and thymus indicating that WS extract stimulated the production of immune cells.
Davis and Kuttan (Citation1998) studied intestinal villus architecture of mice treated with different levels of cyclophosphamide in the presence and absence of WS. Results showed that crypts of the WS-treated group were not fused or any reduction in the number was seen. Also, Villi looked slightly shortened, not distorted, and there was no necrosis of mucous cells throughout the villi. They pointed out that WS is a potent growth factor stimulating the immune system and thereby positively affecting the structure and function of epithelial cells in all parts of villi. It is possible that the change in villus width may be a reflection of differences in immune response. Dibner et al. (Citation2008) reported that Lamina propria is the largest determinant of villus width and is populated with fibroblasts, endothelial cells, lymphocytes, macrophages, and Ig-secreting plasma cells. These results are reflected in primary antibody response findings as evident from the increased IgG. The addition of 150 mg/kg WS to negative control diets significantly increased IgG and villus width. The results indicate that these effects can provide some insight into the relation between immune status and intestinal morphologic parameters.
5. Conclusion
Under the conditions of this study, it was concluded that administration of 150 mg/kg WS and WC significantly increased the production of IgG specific to SRBC on primary and secondary antibody response, respectively. Significant increases in the spleen weight were noted with supplementation of 150 mg/kg WS and 1,25(OH)2D3 at 42 days of age. Also, administration of WS significantly increased villus width at this age. Our results justify further research in this area to determine the optimal dietary inclusion level and the mechanism of action of plant extracts warrants further investigation.
Disclosure statement
No potential conflict of interest was reported by the authors.
References
- Abouzid SF, El-Basuony AA, Nasib A, Khan S, Qureshi J, Choudhary MI. 2010. Withaferin A production by root cultures of Withania coagulans. Int J Appl Res Nat Prod. 3:23–27.
- Alba M, Esmaeilipour O, Mirmahmoudi R. 2015. Effects of Withania coagulans fruit powder and vitamin C on growth performance and some blood components in heat stressed broiler chickens. Livest Sci. 173:64–68. doi: 10.1016/j.livsci.2015.01.001
- Aslam SM, Garlich JD, Qureshi MA. 1998. Vitamin D deficiency alters the immune responses of broiler chicks. Poultry Sci. 77:842–849. doi: 10.1093/ps/77.6.842
- Atta-ur-Rahman Dur-e-Shahwar Naz A, Choudhary MI. 2003. Withanolides form Withania coagulans. Phytochemistry. 63:387–390. doi: 10.1016/S0031-9422(02)00727-6
- Beal RK, Wigley P, Powers C, Hulme SD, Barrow PA, Smith AL. 2004. Age at primary infection with Salmonella enteric serovar Typhimurium in the chicken influences persistence of infection and subsequent immunity to re-challenge. Vet Immunol Immunop. 100:151–164. doi: 10.1016/j.vetimm.2004.04.005
- Berridge MJ, Bootman MD, Roderick HL. 2003. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Bio. 4:517–529. doi: 10.1038/nrm1155
- Cantorna MT, Zhu Y, Froicu M, Wittke A. 2004. Vitamin D status, 1,25-dihydroxyvitamin D3, and the immune system. Am J Clin Nutr. 80; Suppl 6:1717S–1720S.
- Corrier DE, DeLoach JR. 1990. Evaluation of cellmediated, cutaneous basophil hypersensitivity in young chickens by an interdigital skin test. Poultry Sci. 69:403–408. doi: 10.3382/ps.0690403
- Davis L, Kuttan G. 1998. Suppressive effect of cyclophosphamide-induced toxicity by Withania Somnifera. J Ethno Pharmacol. 71:193–200. doi: 10.1016/S0378-8741(99)00206-8
- Davis L, Kuttan G. 2000. Immunomodulatory activity of Withania Somnifera. J Ethno Pharmacol. 62:209–214. doi: 10.1016/S0378-8741(98)00039-7
- Dewir YH, Chakrabarty D, Lee SH, Hahn EJ, Paek KY. 2010. Indirect regeneration of Withania somnifera and comparative analysis of withanolides in in vitro and greenhouse grown plants. J Plant Biol. 54:357–360. doi: 10.1007/s10535-010-0063-6
- Dibner JJ, Richards JD, Knight CD. 2008. Microbial imprinting in gut development and health. J Appl Poultry Res. 17:174–188. doi: 10.3382/japr.2007-00100
- Eerola E, Veromaa T, Toivanen P. 1987. Special features in the structural organization of the avian lymphoid system. In: Toivanen A, Toivanen P, editors. Avian immunology: basis and practice. Boca Raton, FL: CRC Press; p. 9–22.
- Feske S. 2007. Calcium signalling in lymphocyte activation and disease. Nat Rev Immunol. 7:690–702. doi: 10.1038/nri2152
- Garlich JD, Qureshi MA, Ferket PR, Aslam SM. 1992. Immune system modulation by dietary calcium. Proceedings of the 19th World Poultry Congress, Amsterdam, The Netherlands; p. 99–106.
- Gupta SK, Dua A, Vohra BP. 2003. Withania somnifera (Ashwagandha) attenuates antioxidant defense in aged spinal cord and inhibits copper induced lipid peroxidation and protein oxidative modifications. Drug Metabol Drug Interact. 19:211–222. doi: 10.1515/DMDI.2003.19.3.211
- Kean RP, Lamont SJ. 1994. Effect of injection site on cutaneous basophil hypersensitivity response to phytohemagglutinin. Poultry Sci. 73:1763–1765. doi: 10.3382/ps.0731763
- Khan S, Malik F, Suri KA, Singh J. 2009. Molecular insight into the immune up-regulatory properties of the leaf extract of Ashwagandha and identification of the Th1 immunostimulatory chemical entity. Vaccine. 27:6080–6087. doi: 10.1016/j.vaccine.2009.07.011
- Lepage KT, Bloom SE, Taylor JrRL. 1996. Antibody response to sheep red blood cells in a major histocompatibility (B) complex aneuploid line of chickens. Poultry Sci. 75:346–350. doi: 10.3382/ps.0750346
- Leyon PV, Kuttan G. 2004. Effect of Withania somnifera on B16F-10 melanoma induced metastasis in mice. Phytother Res. 18:118–122. doi: 10.1002/ptr.1378
- Liljequist BL, Brake J, Davidian M. 1992. Evaluation of the interdigital skin test for cutaneous basophil hypersensitivity in female broilers. Poultry Sci. 71; Suppl 1:58. (Abstr.)
- Malik F, Singh J, Khajuria A, Suri K, Satti N, Singh S. 2007. A standardized root extract of Withania somnifera and its major constituent withanolide-A elicit humoral and cell-mediated immune responses by up-regulation of Th1-dominant polarization in BALB/c mice. Life Sci. 80:1525–1538. doi: 10.1016/j.lfs.2007.01.029
- Mesaik MA, Haq Zu, Muradb S, Ismail Z, Abdullah NR, Gill HK, Atta-ur-Rahman H, Yousaf M, Siddiqui RA, Ahmade A, Choudhary MI. 2006. Biological and molecular docking studies on coagulin-H, Human IL-2 novel natural inhibitor. Mol Immunol. 43:1855–1863. doi: 10.1016/j.molimm.2005.10.020
- Mirakzehi MT, Kermanshahi H, Golian A, Raji AR. 2013. The effects of dietary 1, 25-dihydroxycholecalciferol and hydroalcoholic extract of Withania somnifera root on bone mineralisation, strength and histological characteristics in broiler chickens. Brit Poultry Sci. 54:789–800. doi: 10.1080/00071668.2013.850469
- Negulescu PA, Shastri N, Cahalan MD. 1994. Intracellular calcium dependence of gene expression in single T lymphocytes. Proceedings of the National Academy of Sciences. USA 91:2873–2877.
- Norman AW. 2008. From vitamin D to hormone D: fundamentals of the vitamin D endocrine system essential for good health. Am J Clin Nutr. 88:1455–1456. doi: 10.3945/ajcn.2008.27049
- Panwar J, Tarafdar JC. 2006. Distribution of three endangered medicinal plant species and their colonization with arbuscular mycorrhizal fungi. J Arid Environ. 65:337–350. doi: 10.1016/j.jaridenv.2005.07.008
- Peelen E, Knippenberg S, Muris AH, Thewissen M, Smolders J, Tervaert JW, Hupperts R, Damoiseau J. 2011. Effects of vitamin D on the peripheral adaptive immune system: a review. Autoimmun Rev. 10:733–743. doi: 10.1016/j.autrev.2011.05.002
- Prasad SK, Kumar R, Patel DK, Hemalatha S. 2010. Wound healing activity of Withania coagulans in streptozoto-cin-induced diabetic rats. Pharm Biol. 48:1397–1404. doi: 10.3109/13880209.2010.486837
- Ross. 2007. Ross 308 broiler: nutrition specification. Aviagen, Scotland, UK; [cited 2009 May 25]. Available from: http://www.aviagen.com/
- SAS. 2003. SAS 9.1. Cary, NC: SAS Institute.
- Shinki T, Takahashi N, Kadofuku T, Sato T, Suda T. 1985. Induction of spermidine N1-acetyltransferase by 1α, 25-dihydroxyvitamin D3 as an early common event in the target tissues of vitamin D. J Biol Chem. 260:2185–2190.
- Shinki T, Tanaka H, Takito J, Yamaguchi A, Nakamura Y, Yoshiki S, Suda T. 1991. Putrescine is involved in the vitamin D action in chick intestine. Gastroenterology. 100:113–122. doi: 10.1016/0016-5085(91)90590-H
- Shohat B, Kirson I, Lavie D. 1978. Immunosuppressive activity of two plant steroidal lactones withaferin A and wi-thanolide E. Biomedicine. 28:18–24.
- Silberman DM, Wald M, Genaro, AM. 2002. Effects of chronic mild stress on lymphocyte proliferative response. Participation of serum thyroid hormones and corticosterone. Int Immunopharmacol. 2:487–497. doi: 10.1016/S1567-5769(01)00190-4
- Tabor CW, Tabor H. 1984. Polyamines. Annu Rev Biochem. 53:749–790. doi: 10.1146/annurev.bi.53.070184.003533
- Tahmasbi AM, Mirakzehi MT, Hosseini SJ, Agah MJ, Kazemi Fard M. 2012. The effects of phytase and root hydroalcoholic extract of Withania somnifera on productive performance and bone mineralisation of laying hens in the late phase of production. Brit Poultry Sci. 53:204–214. doi: 10.1080/00071668.2012.662628
- Wang D, Li X, Xu L, Hu Y, Zhang B, Liu J. 2006. Immunologic synergism with IL-2 and effects of cCHMIs on mRNA expression of IL-2 and IFN-γ in chicken peripheral T lymphocyte. Vaccine. 24:7109–7114. doi: 10.1016/j.vaccine.2006.07.005
- White RG, Henderson DC, Eslami MB, Neilsen KH. 1975. Localization of a protein antigen in the chicken spleen. Effect of various manipulative procedures on the morphogenesis of the germinal centre. Immunology. 28:1–21.
- Yamauchi K, Buwjoom T, Koge K, Ebashi T. 2006. Histological alterations of the intestinal villi and epithelial cells in chickens fed dietary sugar cane extract. Brit Poultry Sci. 47:544–553. doi: 10.1080/00071660600963149