During intensive poultry breeding, birds subject to diverse stressors as vaccination, hypoxia, starvation and infection; these stress conditions, activate the hypothalamic-pituitary-adrenal (HPA) axis, leading to an increase in concentration of circulating corticosterone and altered cellular immune responses1-3. Interestingly, animals have defense systems which protect them from various stresses, one of which is expression of heat shock protein (HSP). Heat shock protein70 (HSP70) is one of the most well-known HSPs. It is a 70-kDa protein expressed in almost all organs4 such as the gastrointestinal tract5 and the nervous system6. When the organism is subjected to highly stressful conditions, it could systemically enhance the induction of heat shock protein (HSPs) to protect the body7. It has been reported that, HSP70 shows apoptosis-suppressive effects8 and anti-inflammatory activity9, leading to cytoprotective functions from various stresses. Dietary modulations are one of the proven approaches to minimize the negative effects of stress on production and anti-oxidant variables in chicken. Feed additives from plant origin are gaining wider acceptability in recent times both in human health practices and livestock production owing to several benefits associated with the natural compounds. Essential oils (phyto-plus powder) is a mixture of herbal extracts, including, Thyme, Oregano, peppermint, Garlic, and Rosemary. Origanum and Thymus, are regarded as oil-rich on the basis of their essential oil content >2%10. The major constituents of the oils of thyme and oregano species have been reported to be thymol, carvacrol and c-terpinene11,12.
In general, thymol [5-methyl-2-(1-methylethyl) phenol], is a main component of thyme essential oil, and its isomer and carvacrol [2-methyl-5-(1-methylethyl)phenol], is a main component of oregano essential oil13. Thyme ingredients seemed to protect against peroxide- and mutagen-induced DNA damage in human lymphocytes14,15. Carvacrol dietary supplementation has pharmacological effects and beneficial health effects, such as antimicrobial, antioxidant, anticancer, antiplatelet, antiviral, anti-inflammatory, antifungal, anti-Alzheimer disease, anti-obesity activity and growth promoting properties16. Wieten et al.17 recorded that Carvacrol, had a notable capacity to co induce cellular HSP70 expression in vitro and upon intra gastric administration, in Payer's patches of mice in vivo. Murakami et al.18 reported that adding essential oil to the broiler’s ration increased villus height in the jejunum on day 14. Phytogenic feed additive may also reduce mucosal thickness, thus contributing to the diffusion of nutrients to the apical surface of epithelial cells and increased absorption and feed efficiency19. Furthermore, in the C. perfringens challenged broiler chickens, Du et al.20 reported that the dietary supplementation of essential oils, which contained 25% thymol and 25% carvacrol as active components could alleviate the intestinal injury by improving intestinal integrity and modulating immune responses. In contrast, it was reported that supplementing essential oils to the broiler’s diets had no effect on villus height in the jejunum statistically, but it increased villus height numerically21.
Aim of the study: This study aimed to evaluate the effect of essential oils (Phyto-plus) as antioxidants and antistresser in broiler chicken by measuring, corticosterone, Heat shock protein (Hsp70), superoxide dismutase, glutathione peroxidase, catalase (CAT) and malondialdehyde (MDA), in addition to employment histopathological examination of the liver, kidney and intestine .
MATERIALS AND METHODS
Phyto-plus powder is a dry stabilized preparation manufactured by (Kanzy Medipharm, Canada), it is a mixture of herbal extracts (Garlic, Thyme, Oregano, Peppermint, Rosemary) with Calcium carbonate as carrier.
Experimental design: The trial was carried out at Poultry Production Farm, Feed Research House, Orabi Community, Qalyubia Governorate. A total of 180 one-day old Ross 308 broiler chicks (as hatched) with an initial body weight around 44 g were obtained from a local commercial hatchery. The chicks were weighed and randomly distributed in 3 pens 60 chicks each, to examine the effect of 2 experimental treatments comprising three feeding regimens: (GP1) Basal diet without Phyto-plus powder supplement (control group) and basal diet supplemented with either 100 or 400 g Phyto-plus powder per ton of ration (GP2 and GP3, respectively) for 6 weeks.
All the experimental birds were reared in well ventilated shed on soft wood shaving litter used as bedding material in pens. Basal starter (0-14 day), grower (15-24 day) and finisher (25- end) diets were formulated according to the nutritional recommendation of National Research Council22 for broiler. Feed and water were available ad-libitum during the experiments.
Growth performance: Chicks of all groups were weighed individually before the start of the experiment and at the end of the 3rd and 6th week of experiment.
Blood sampling: Blood samples were collected from each bird via heart puncture at the end of the 3rd and 6th week. Blood samples were taken without anticoagulant in a clean and dry centrifuged tube, left to clot at room temperature and then centrifuged at 3000 rpm for 15 min. The sera were collected for analysis of all biochemical parameters and Oxidant/antioxidant markers.
Tissue samples: Specimens from liver, kidney and intestine were collected from slaughtered birds for histopathological examination.
Biochemical analysis: The biochemical assays of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were estimated according to Reitman and Frankel23. Total Protein was measured according to Doumas, et al.24, Uric acid was measured according to Yound25 and serum creatinine was measured according to Kroll26.
Oxidant/antioxidant markers including Malondialdehyde (MDA)27, Catalase (CAT)28 and Superoxide dismutase (SOD)29 were calorimetrically assayed using chemical kits (Biomed Egypt) and Shimadzu UV 240. Glutathione peroxidase (GST PX) was measured by using ELISA Kits cat. No. MBS 1600242. Heat shock protein 70 (HSP70) was estimated by using ELISA Kit cat. No. MBS035085. Corticosterone hormone was determined by using ELISA Kit cat. No: MBS761865.
Histopathological examination: The intestine, liver and kidney samples from birds were taken for each treatment after 3 and 6 weeks of phyto-plus supplementation. Samples were cleaned with distilled water to remove the intestinal contents and fixed in 10% neutral buffered formalin solution then dehydrated, cleared and embedded in paraffin wax, then sectioned to 4-5 micron thickness, prepared and stained with Hematoxylin and Eosin stain (H&E) and examined microscopically30. Morphometric measurements of the intestinal villi were performed including villus length and villus width. Measurements of 10 complete villi for villus length and width per sample of each bird intestinal section were performed at 40 magnifications using Image J Analyzer software31 and the average of these values were used for statistical analysis.
Statistical analysis: The obtained data were computerized and analyzed for significance, standard error and variance was calculated using SPSS 1432.
The effects of two doses of Phyto-plus Powder on the live body weight and body weight gain of broiler chicken are presented in Table 1. There were no significant differences in the live body weight of chickens among groups (control group, Phyto-plus 100 and Phyto-plus 400) after 3 weeks. However, after 6 weeks of Phyto-plus Powder supplementation the birds showed a significantly increased live body weight in GP2 as compared with the control group, while the increase in the live body weights were non-significant in GP3 compared to control group. Chicken fed diet supplemented with 100 or 400 mg of Phyto-plus t1 of ration exhibited a higher body weight gain after 3 and 6 weeks of Phyto-plus supplementation, that was not statistically different in birds with high dose of Phyto-plus compared to the control group.
Table 2 exhibits the level of heat shock protein 70 (Hsp70) and corticosterone hormone in chickens fed on phyto-plus at dose of 100 or 400 g t1 of ration for 6 weeks. The result demonstrated elevation in HSP70 levels significantly in two treated groups of chickens after 3 weeks and in chickens with low dose (GP2) of phyto-plus, whereas, the corticosterone hormone lowered significantly in the two treated groups of chickens (GP2 and GP3) as compared to the control one.
Concerning, antioxidant enzymes activity, the results revealed a significant elevation in the activities of CAT, SOD and GH-PX levels of birds in Group 2 after 3 and 6 weeks of phyto-plus supplementation, while these enzymes increased significantly in chickens fed diet supplemented with high dose of phyto-plus only after 6 weeks as compared to the control group. There were no significant changes in MDA level in chickens of the two treated groups, although its level decreased non-significantly with low dose of phyto-plus compared to the control while Gp3 (400 mg phyto-plus t1 of ration) revealed significant increase in AST activity and creatinine concentration as compared to other groups indicating that the low dose of phyto-plus (100 g t1 of ration) is more safe than its high dose (Table 3).
Liver and kidney markers were measured for the analysis of the safety use of phyto-plus (Table 4). The results demonstrated that birds fed on phyto-plus at dose of 100 g t1 of ration (Gp2) showed normal values of ALT, AST, uric acid and creatinine with a significant increase in total protein level after 3 and 6 weeks of supplementation as compared to the control, while Gp3 (400 g phyto-plus t1 of ration) revealed significant increase in AST activity and creatinine concentration as compared to other groups. Indicating that the low dose of phyto-plus (100 g t1 of ration) is safer than its high dose.
The results of villus height and width of the control and experimental groups are presented in Table 5. Results showed that supplementation of broilers diets with 100 g t1 ration in group two after 3 weeks revealed proliferation in glandular lining epithelial cells (Fig. 1) and increased the length (1043±44.96) and width (182±9.02) of the intestinal villi (Fig. 2). Histological analysis of liver and kidney did not indicate any pathological changes but appear normal (Fig. 3 and 4). After 6 weeks the villi showed hyperplasia of the goblet cells in addition to increase the length (1057.10±13.74) and width (163.90±5.09) of villi compared to 3 weeks (Fig. 5 and 6), the villi were found to be very long in this group.
Concerning the intestine, liver and kidney of chickens in group 3 which fed diet supplemented with phyto-plus at dose of 400 g t1 of ration for 3 weeks, the liver revealed widely dilated central vein (Fig. 7) and kidney showed hydropic degeneration in the epithelial cell lining of renal tubules (Fig. 8) but after 6 weeks the intestinal villi revealed slightly decrease in the length (996.50±24.52) and width (149.30±9.32) compared to control group (Fig. 9) while n liver and kidney the lesions progressed to fatty degeneration (signet ring appearance of hepatocytes) (Fig. 10) and tubular nephrosis with cystic dilatation of some renal tubules (Fig. 11).
Thymol and carvacrol are believed to exhibit a range of beneficial physiological effects. The obtained results showed that chickens fed diets supplemented with low dose of Phyto-plus Powder (Gp2) exhibited a significant heavier live body weight (LBW) at 6 weeks and body weight gain (BWG) at 3 and 6 weeks of ages. In consistent with these results, Ocak et al.33 recorded a significant higher LBW at 21 and 42 day of ages as well as higher BWG from 7-35 day of age in broilers fed peppermint and thyme. Moreover Al-Kassie34 and Abid35 showed that chickens fed with thyme supplement had significantly heavier live body weight and body weight gain. Furthermore, using oregano herbs tested in broiler diets improved body weight and feed intake at 1,000 ppm36, 500 ppm37, 100 and 250 ppm38. These may be due to the fact that thyme improves diet palatability as well as stimulates and improves the appetite and the digestive process39. On the another hand, Cross, et al.40 reported that using 5 g kg1 thyme caused a substantial decrease in BWG approaching almost a level of significance.
It is well known that heat shock protein 70 (HSP70) provides protection against free radical toxicity therefore; it is involved in the protection of stressed cells and organisms due to its prominent response to diverse stressors41. The current study was performed to identify the enhanced HSP70 in response to phyto-plus and its antistress effect. The obtained results showed a significant increase in the levels of HSP70 with a significant decrease in the levels of corticosterone hormone in groups of birds fed diet supplemented with phyto-plus at dose of 100 g t1 of ration for 3 and 6 weeks.
In accordance with these results Wieten et al.17 found enhanced cellular expression of HSP70 in Payer's patches of mice after intragastric administration of carvacrol, attributing to boosting of cellular HSP70 levels in Payer's patches which led to increase in presentation of HSP70 peptides on antigen presenting cells. Furthermore, Ito et al.42 recorded that human daily consumption of enzyme-treated asparagus extract for 7 days increased expression of HSP70 mRNA. It has been reported that HSP70 mRNA expression were higher in chicken hearts under heat stress43 and in quails exposed to cold stress41, indicating that HSP70 plays a critical role in cellular homeostasis44 and protects from stress sensitivity through the oxidant defensive anti-inflammatory and other mechanism42. Intriguing, the increased HSP70 has been shown to protect skeletal muscle cells against oxidative stress and free radical toxicity45. Since elevation of HSP70 levels leads to cell protection and results in protecting organs against various stresses, the antistress activity of phyto-plus was established. Additionally, lowering the levels of stress hormone as corticosterone hormone in phyto-plus treated groups in the present study is considered another hypothesis sustained the antistress effect of phyto-plus. This may be due to the fact that the autonomic nervous, endocrine, and immune systems interact with complicated biological defense reactions (as heat shock proteins) against various stress42. Therefore, it may be due to the fact that enhanced HSPp70 by phyto-plus had direct inhibitory effect on endocrine gland (adrenal cortex) secreting corticosterone. Further studies were warranted to elucidate validity of this hypothesis through the effect of phyto-plus on HSP70 and corticosterone in stressed boiler chickens.
The present data exhibited increased antioxidant enzymes activities (superoxide dismutase, catalase and glutathione peroxidase) in birds fed diet supplemented with phyto-plus for 6 weeks. The results are in agreement with the results of Hashemipour, et al.46 who showed that thymol plus carvacrol supplementation linearly increased superoxide dismutase and glutathione peroxidase activities and decreased malondialdehyde level in thigh muscle of chickens at d 42 and serum and liver at d 24 and 42, suggesting that the high antioxidant activity of these herbs are due to the presence of phenolic OH groups that act as hydrogen donors to the proxy radicals produced during the first step in lipid oxidation, thus retarding the hydroxyl peroxide formation. Based on these findings, the authors recorded that thymol and carvacrol might play an important role as an exogenous antioxidant and in turn confirms the antioxidant activity of phyto-plus. Moreover, Silveira et al.47 recorded that, herbs rich in flavonoids such as thymol and carvacrol could improve the immune functions through acting as antioxidants and extending the activity of vitamin C. Furthermore, luna et al.48showed that antioxidant activity of both thymol and carvacrol feed supplementation have similar effectiveness to retard lipid oxidation compared to the supplementation with the antioxidant butylated hydroxyl toluene, indicating that supplementation with natural antioxidant thymol and carvacrol could be applied to improve poultry meat quality.
Morphological changes in the gastrointestinal tract caused by phytogenic feed additive (PFA) may provide further information on possible benefits to the digestive tract. In general, PFA revealed significantly increased in the villus height (VH) across the small intestine. In the absence of any inflammation, this may be due to increase absorptive surface area and efficiency of digestion and absorption49. A similar effect of PFA on VH has been reported by Namkung et al.50. In the present experiment Supplementation of broilers diets with 100 g phyto-plus t1 of ration for 3 weeks increase the length and width of the villus in the duodenum and proliferation of glandular lining epithelial cells. The increase of villi length is an indicator of increased intestinal absorption surface area. Similar to this result, Murakami et al.18 reported that adding essential oil to the broiler’s ration increased villus height in the jejunum on day 14. In contrast, Jamroz et al.21 found that supplementing essential oils to the broiler’s diets had no effect on villus height in the jejunum statistically but it increased villus height numerically. Increasing the villus height in the duodenum villi could increase the efficiency of absorptive process considering the fact that the majority of absorption occurs in the duodenum. Furthermore, greater villus height increases the activities of mucosal digestive enzymes, resulting in improved digestibility49,51. As intestinal crypts are the sources of epithelial cells for villi.
Histological analysis of liver, kidney did not indicate any pathological changes but appear apparently normal with low dose of phyto-plus, indicating that thymol as a potential antioxidant, protects cells against damage by lowering/preventing the loss of mitochondrial membrane potential and inhibition of reactive oxygen species overproduction52. Increased length and width of villi with numerous proliferation of goblet cells after 6 weeks of phyto-plus supplementation, in this study, are in agreement with the results obtained by Murugesan et al.49 who reported that villus height, width and goblet cell increased with increase of age of the broiler chicken. This shows a promising effect of phyto-plus on gut health of broilers. The increased villus height and villus width are linked with increased surface area which, in turn, leads to better digestion and absorption. Similarly, Chacher et al.53 observed that a higher number of goblet cell per villus indicates higher production of mucins and glycoprotein compounds that bind with the pathogenic bacteria thus preventing their attachment with the intestinal mucosa. It is worth to mention that essential oils have the ability to reduce the growth of pathogenic bacteria and increase the numbers of benefit one when fed to birds54. On the contrary, Vieira et al.55 reported that inclusion of some organic acids did not significantly influence the villus height at any age of broiler’s life.
Regarding to pathological changes in intestine, liver and kidney of chickens fed diet supplemented with phyto-plus at dose of 400 g t1 of ration, showed slightly decrease in length and width of the intestinal villi, in addition to the lesion of liver and kidney progress after 6 weeks to fatty change and coagulative necrosis in epithelial cells lining tubules. The results of the present study were in line with the findings of Yigit et al.56 who reported that high level of oregano oil exerted slightly negative effects on organ histology as slightly degeneration and necrosis of liver and kidney of fish, this may be related to toxic constituents, excessive doses, or allergic conditions but they generally have no effects on health when used in the proper doses. Additionally, Fernandez et al.57 found sinusoidal dilatation, vascular congestion and fatty degeneration which observed in all liver of quail fed diet supplemented with 4-5 g thymol per kg of feed, indicating to over feeding. The obtained result in Table 4 and 5 also confirmed that the low dose of phyto-plus (100 g t1 of ration) is more effective and safe than its high dose (400 g t1 of ration).
Phyto-plus play an important role as antioxidant and antistress. It also improved bird growth performance and the intestinal histomorphology. Based on the results obtained in this study it can be concluded that the low dose of phyto-plus (100 g t1 of ration) was an optimum supplementation dose compared to the high dose (400 g t1 of ration). More studies are needed to explore its capabilities to support this study.