Effects of dietary supplementation of Amla, Chicory and Leek extracts on growth performance, immunity and blood biochemical parameters of broilers

Abstract The need for solutions to relieve heat stress and its undesirable effects on broiler production increased, especially in hot climatic areas. The antioxidant and immune stimulant effects of herbal extracts of Emblica officinalis (Amla), Cichorium intybus (Chicory) and Allium porrum (Leek) were examined against the adverse effects of heat stress in broilers. One thousand Cobb 500 one-day-old chicks were used for this experiment. Birds were divided into four equal groups. The first served as the control group. The second received Amla extract (3 g/kg diet), the third received Chicory extract (3 g/kg diet) and the last group received Leek extracts (3 g/kg diet). All groups were daily subjected to heat stress for 12 h by increasing house temperature to 8 ± 2 °C (10:00 am to 10:00 pm daily) using an electric heater, while relative humidity was kept at 47 ± 3%. Results revealed significant improvement in final body weight in treated groups, especially the Chicory and Amla-fed groups. Total weight gain was improved by dietary supplementation with herbal extracts, while feed intake, feed conversion ratio (FCR) and mortality percentage were reduced. The positive impact of the herbal extracts was also clear on carcase traits in the treated groups. Meat and serum lipids were significantly reduced in treated groups. White blood cells, phagocytic index and antioxidant enzyme levels were positively improved by dietary supplementations. In conclusion, using Amla, Chicory and Leek extracts positively improved broilers’ growth performance, immunity and oxidative stress status under heat stress conditions. Highlights Heat stress causes huge economic losses in livestock production worldwide. Herbal extracts are rich in antioxidants at low prices than other feed additives. Herbal extracts can enhance the defense system and reduce the negative effects of heat stress.


Introduction
Heat stress is one of the poultry industry's common causes of high economic losses (Abo Ghanima 2020).It results in reduced growth performance due to reduced feed intake as a sequence of appetite loss (Awad et al. 2020).The negative effects of heat stress may increase mortalities and, in progressive cases, may result in very high losses.The effects of heat stress occur as a result of the changes and modifications inside a bird's body, such as the elevation of blood alkalosis due to excessive loss of carbon dioxide as a result of increased respiratory rate during heat stress to help birds to lose excessive body temperature (Barrett et al. 2019).
Gut status during heat stress seems to greatly affect the integrity of enterocyte cell walls, causing easy way for pathogenic microbe's invasion and that ends with enteritis and loss of a lot of nutrients due to lowering digestibility and absorption (Liu et al. 2016).Besides oxidative stress, which resulting in the disruption of cells and interfering with their functions, creating adverse challenges for the immune system (Altan

Materials and methods
All procedures in the current study were approved by the Local Experimental Animal Care Committee and the Institutional Ethics Committee of the Animal Husbandry and Animal Wealth Development Department, Faculty of Veterinary Medicine, Damanhour University, Egypt (DMU/VetMed-2019-/0145).

Experimental birds, design and management
A total of 1000 one-day-old Cobb 500 chicks (Obtained from El Wataniya Hatcheries, Km 56 Cairo -Alexandria desert road -Alexandria -Egypt) were used for the study.The birds were subdivided into four equal groups, each of 250 birds.The first treatment served as the control group.The second received with Amla extract (3 g/kg diet) (Manufactured by Till and Yield exports private LTD -Madhya Pradesh -India), the third received Chicory extract (3 g/kg diet) (Manufactured by Shaanxi Hongda Phytochemistry Co., Ltd.-Shaanxi -China) and the last group received Leek extract (3 g/kg diet) (Manufactured by Shaanxi Qinglan Bio-Technology Co., Ltd.-Shaanxi -China).The chemical composition of herbal extracts is shown in Table 1.All groups were subdivided into five equal replicates (n ¼ 50).All the birds brooded at 33 C, then decreased by 3 C weekly till it reached 24 C and then maintained at that rate.All groups were daily subjected to heat stress for 12 h by increasing the house temperature to 8 ± 2 C (10:00 am to 10:00 pm daily) using an electric heater, while relative humidity was kept at 47 ± 3%.The composition of basal diet is shown in Table 2.

Productive performance and carcase traits
Per replicate, body weight and total feed consumption of day-old, 21-day-old and 42-day-old birds were determined.By deducting day-old weight from 42day-old weight, total body weight gain (daily weight gain) was estimated.Total feed divided by total gain was used to calculate total feed efficiency.Birds were weighed after a 12 h of fasting and before being slaughtered.Five birds from each replicate were wetly plucked and eviscerated.Internal organs (liver and gizzard, heart and spleen) were individually weighed to determine their weights and dressing percentage.Offal was defined as the blood, viscera, lungs, legs, head and neck, and it was thrown away.The carcase was weighed after all of the abdominal fat in the pelvic and abdominal cavities had been removed.The three cuts from the carcase were the left fillet (the deskinned left breast muscle on the left side of the sternum), the weight of two average thighs and the breast (breast muscles with the sternum).Each replicate's total mortality for the whole week was noted.

Blood biochemistry
Two blood samples from five birds from each replicate were taken at 42 days into centrifuge tubes.The first tube contained a 3.2% sodium citrate solution used to measure the activity, phagocytic index and the number of leucocytes.After being given time to clot, the other tube was centrifuged at 4500 xg for 15 min.The serum samples were obtained and kept in a deep freezer at À20 C until being analysed.The phagocytic activity and index were calculated using the method described by Kawahara et al. (1991).Following the manufacturer's instructions, spectrophotometric measurements of total lipids (TL), triglycerides (TG, Fossati and Prencipe 1982), total cholesterol (TC), urea (Fawcett and Scott 1960), lactate dehydrogenase (LDH, Song et al. 2016), creatinine (Schirmeister 1964), Alanine Aminotransferase (ALT, Reitman and Frankel 1957) and Aspartate Aminotransferase (AST, Reitman and Frankel 1957) were made using commercial kits from Biodiagnostic Co., Egypt (Spectronic 1201; Milton Roy, Ivyland, PA, USA).
Using the ELISA Kit from Quanti ChromTM, BioAssay Systems and Cayman Chemical Company, USA, the activities of oxidative stress markers such as malondialdehyde (MDA, Jo and Ahn 1998), glutathione peroxidase (GPx, Kokkinakis and Brook 1979), superoxide dismutase (SOD, Sun et al. 1988), catalase District, Wuhan, Hubei Province, China).We followed the manufacturer's instructions for each step.Utilising BiodiagnosticV R kits, the concentrations of alanine, ALT, AST, creatinine and urea were analysed (Biodiagnostic -Giza -Egypt).For each step, we did as the manufacturer instructed.The uric acid analysis was carried out using the acid test kit from Sigma-uric Aldrich.

Tissues biochemistry
Folch et al. (1957) method for gathering meat samples, storing them and preparing the extract was followed.Meat triacylglycerol was calculated from triglycerides, and meat cholesterol level was assessed using a particular Biodiagnostic V R test.

Statistics
Data were analysed using the general linear model and one-way analysis of variance in SPSS software version 20.0 with the following model: Where: X ij ¼ An observational data; m Overall mean; A j Effect of treatment; e i Random error.

Growth performance
Table 3 inferred significant improvement in 21-day and final body weights in treated groups, especially Chicory and Amla groups.Similarly, total weight gain improved by treatment, but FI, FCR and mortality rate reduced by treatments compared with the control group.
These findings concur with those reported by Izadi et al. (2013), who found that feeding Chicory extract to broilers increased their BW% but decreased their FCR.These results specifically conflict with those of Khoobani et al. (2019).They found no significant (p > .05)effects of dietary Chicory powder on FI during broilers' first and second growing periods.In addition, birds fed diets containing 0.10% Chicory throughout the entire experiment (1-42 days) had significantly higher feed intake (p > .05)than birds fed 0.15%-0.20%Chicory.
When Chicory extract was added to the birds' diets, their feed utilisation was much lower than that of the control group, which is consistent with recent findings (Faramarzzadeh et al. 2017).Additionally, chickens fed various herbal plants showed a significant improvement in FCR, according to Cabuk et al. (2006).Furthermore, in line with our findings, Liu et al. (2011) discovered that adding Chicory powder to the diet significantly improved FCR for the first 13 days of the feeding period.Broilers fed diets containing Chicory extract may exhibit higher development performance due to the superior length, number and surface area of intestinal villi compared with an increased digestive and absorptive capacity of the jejunum (Mroz et al. 2005;Izadi et al. 2013).
In broiler chicks, Chicory powder extracts improved gut microbiota and growth performance (Khoobani et al. 2019).Chicory roots contain inulin, while the leaves contain phosphorus, magnesium, potassium and a bitter glycoside called shikonin (Nandagopal and Kumari 2007).Inulin and oligofructose enhance gastrointestinal absorption of minerals such as calcium, magnesium and iron (Yeung et al. 2005).Abrams et al. (2007) stated that the addition of Chicory roots to the diet improved growth performance.Our results concur with those of Kashmiri et al. (2020), who found significant (p > .05)differences in body weight between the Amla powder-fed groups and the control group.Still, no such changes were found in BWG when Amla powder was used in the first, second or third weeks of age.The findings obtained by Ghavate et al. (2009), Shivaji (2012), Patel et al. (2016) and Tiwari et al. (2016) were all comparable.The ascorbic acid, gallic acid and tannic acids found in Amla may have an antioxidant and anabolic impacts.Amla supplementation at 0.4 and 0.8% improved BWG in broilers compared to the control group (Patel et al. 2016).Similar effects on broilers were also discovered by Kumar et al. (2012, 2013), and Patil et al. (2014).Sanjyal and Sapkota (2012), Patil et al. (2012) and Gaikwad et al. (2016) reported that broiler chicks in the control group consumed more feed than those in the Amla-treated group.Compared to the broilers that were not supplemented, a linear decrease in FI was seen with increasing levels of supplementation in all broilers that had Amla supplementation (Kumari et al. 2012).
The present study was contrasted with the observed by Kashmiri et al. (2020), who found that the weekly feed intake was higher in Amla group (0.75%) than the control.Authors recommended adding Amla powder at levels of 0.25, 0.50 and 0.75% for the best feed intake.Also, the FCR of broilers improved due to adding Amla powder to the diet compared with the control group.Similar findings were also reported by Ghavate et al. (2009), Patil et al. (2012), Shivaji (2012), Kumar et al. (2013), Gaikwad et al. (2016) and Mandal et al. (2017) in broiler chickens.El-Khabery et al. (2016) inferred that broilers fed diets supplemented with 4%, 6%, 8% and 10% Leek significantly improved total final BW, WBG, TFI and FCR than the control group.These results may be due to the Leek concentration of sulphur elements that are good active antimicrobial agents.The BWG was greater in the chicks whose diet included 3% onion extract supplements (Goodarzi et al. 2014).Aji et al. (2011) revealed that birds fed diets containing garlic and onion gained more body weight than the untreated group.Given that Leek extract has a high concentration of polyphenols, which raises calorie and feed intake, it may improve growth performance and FI (Mahmoud et al. 2009).

Carcase traits
No noticeable changes were found in most carcase features (liver, gizzard, heart, spleen, thigh and shoulder) as a result of treatments (Table 3).Dressing %, Breast % and left filet % significantly increased in the treated groups, while abdominal fat reduced compared to the control group.Khoobani et al. (2019) found no significant variability in carcase traits by adding Chicory powder.However, the percentage of abdominal fat significantly decreased in chicks fed Chicory powder-diet supplemented.Chicory powder or a probiotic combination added to the feed of broilers may improve the overall intestinal microenvironment, reduce endogenous nitrogen loss and reduce the release of abdominal fat in broilers (Panda et al. 2009).Due to adding Chicory powder, there were no significant differences in broilers' internal organs and carcase traits (Mirza Aghazadeh and Nabiyar 2015).Similarly, Mudalal et al. (2020) reported that some carcase traits were not affected by due to herbal extract addition, but the dressing percentage significantly improved compared to the control.In contrast, Gaikwad et al. (2016) found no significant effects on dressing percentage by 0.5% Amla levels, but a higher dressing % was recorded in birds fed 1% Amla supplement.
Our findings are in line with those of El-Khabery et al. ( 2016), who noticed that although broilers given various dietary Leek extracts did not show any differences in the percentages of their liver, heart, stomach or spleen, the dressing percentage was significantly higher in comparison to the control group.These findings are consistent with those of Aji et al. (2011), who reported that carcase features of broilers fed garlic and onion did not differ.
No significant difference in the heart, intestine, spleen and stomach was observed due to Leek leaf extract in comparison to the control group, but carcase dressing % was improved (Al-khalaifah et al. 2020), the improvement in dressing %, particularly in the group that fed Leek, might be due to the increased of final BW that as a result of fed on Leek and reflected on the dressing weight.

Biochemical parameters
Results showed that meat cholesterol, meat triglycerol and total lipids significantly reduced due to herbal extract addition compared to the control group (Table 4).Results of the current study support those of Yusrizal and Chen (2003a), who found that adding Chicory extract to a broiler's diet reduced total cholesterol and triglycerides.Also, Yusrizal and Chen (2003b) found that adding Chicory-based fructans to chicks' diets resulted in a drop in their TC and TG levels.Similar conclusions were reached by Kok et al. (1996), who found that the hypotriacylglycorolemic impact of oligofructose by decreasing hepatic fatty acid and the inhibition of lipogenic enzymes, which results in the formation of triacylglycerol.
Blood samples from Amla-extract groups revealed that cholesterol levels were significantly lower than the control group.This finding suggests that Amla tannin has a variety of minimal effects on cholesterol levels and other types of body fat (Sai Ram et al. 2003).Vidyarthi et al. (2008) reported that Amla supplementation to broiler diet resulted in lower cholesterol.According to El-Khabery et al. (2016), Leek powder at levels of 4%, 6%, 8% and 10% significantly reduced TC compared to the control group.While the 8% level of Leek powder was the most effective, but no significant effects were found on TP, albumin, globulin.

Liver and kidney function
Data in Table 5 show that urea, creatinine, ALT, AST and LDH were significantly reduced due to dietary herbal extracts supplementation.Conversly, Yusrizal and Chen (2003a) and Yusrizal and Chen (2003a) found that adding Chicory extract to broiler's diet did not affect urea levels.El-Khabery et al. (2016) noted that adding Leek powder has no significant effects on ALT, AST, creatinine or urea.No significant effects on serum ALT, AST, creatinine and urea were found in birds fed on Leek extract compared to the control group (Al-khalaifah et al. 2020).

Antioxidant parameters
It is clear that MDA, Catalase, GRP, SAA and TRF were significantly decreased in groups treated with the herbal extracts (Table 6).In contrast, GPx and SOD were significantly improved; the best values were noticed in the Chicory extract group.Gurram et al. (2022) revealed that a significant improvement in the health and oxidative status of the birds might be reversed by the constant state of activity of antioxidant enzymes in all test groups.These findings agree with Juc a et al. ( 2020), who hypothesised that increasing the concentration of antioxidant enzymes in our study may be caused by the free radical removal.According to their findings, broilers fed inulin and Chicory powder had lower lipid peroxidation levels and higher levels of SOD and catalase activities.The improved dominance of intestinal pathogens in the gut may cause the increased antioxidant enzyme activity.In the intestine, aerobic bacteria (Bacillus spp.) use oxygen to create an aerobic environment for anaerobic bacteria to colonise.As a result, lactic acid bacteria create more acidic habitats, which prevent the formation of opportunistic pathogens (Sako et al. 1999).Additionally, compared to the control group, the vitamin C and Amla groups had a significantly lower concentration of AST and ALT, according to Aljumaily et al. (2019).
Similar results were obtained by Maini et al. (2007), who found that antioxidant supplementation (Amla extract, vitamin E and dried mint) dramatically reduced the levels of lipid peroxidation in erythrocytes, as opposed to the control group, which had an increased in lipid peroxidation concentration as well as decreased glutathione activity (GSH) and superoxide dismutase (SOD) activity.In the other case, Mirzaei-Aghsaghali (2012) mentioned that onion could synthesise bile acid and consequently enhance lipid digestion.The latter authors reported that onion tonic increased the secretion of pancreatic enzymes, exceptionally lipase and amylase.As well, Goodarzi and Nanekarani (2014) demonstrated that essential onion oil enhances digestion.

Blood cells
Data in Table 7 show a significant improvement in WBC, phagocytic index, phagocytic activity, eosinophil % and lymphocytes % due to herbal extracts addition, especially Chicory extract.Also, monocytes % improved by Chicory extract only compared with other treatments.Islam et al. (2020) reported that using Amla and Aloe vera as dietary supplements had no significant effects on the measured parameters (RBC, WBC, HCT and Hb).These findings are in line with Mekala (2014), who found no differences in haematological parameter observations between the Aloe vera and Amlasupplemented groups and the control.These results stated that adding Amla and Aloe vera to the diet maintained broilers' health.Thiamine, riboflavin, folic acid and other necessary amino acids are available in Aloe vera gel and Amla, which may encourage the erythropoietic system to make red blood cells.These elements might be beneficial for the immune system's activation as well as the thymus, spleen and bone marrow functions, which are associated with the genesis of blood cells.Also, Amla can improve red blood cells count and protect them and plasma proteins from the reactive oxygen species that induce oxidative damage.The presence of gallic acid and quercetin are Amla's major constituents (Li et al. 2002).et al. (2017) showed no significant differences in haemoglobin (g %), heterophil: lymphocyte ratio or lipid peroxidase enzyme activity (nmol/ml) when different doses of Amla supplements were used.These results disagree with those of Lin et al. (2003) and Abdel Raheem and Ghaffar (2004), who claimed that ascorbic acid and Amla extract supplements, were beneficial to the immune system when taken in the hot summer conditions.It has been demonstrated that adding Amla fruit powder to feed can improve broiler performance and provide antistress, adaptogenic, immunogenic and growth-stimulating advantages (Sapcota et al. 2005;Wadhwa et al. 2007).

Mandal
According to Mosleh et al. (2013), using essential oils improves humoral immune responses and shortens the time until faecal viral shedding occurs in chickens given live Newcastle vaccines.Contrarily, the birds that got both the live Newcastle vaccination and the garlic extract combination showed better immune responses than the group that received only the vaccine, as Garbaa et al. (2013) reported in broiler chicken and by Jahanian and Rasouli (2012) in laying hens.

Conclusions
Finally, it is concluded that using Amla, Chicory and Leek extracts in broiler dietswas beneficial in improving feed conversion, weight gain, dressing%, breast%, biochemical parameters and antioxidant parameters, especially with adding Amla and Chicory extracts under heat stress.

Table 1 .
Chemical composition of herbal extracts.

Table 3 .
Mean ± standard error of the effect of Amla, Chicory and Leek extracts on body weight, total feed intake, total weight gain, total feed conversion, mortality % and carcase traits of broilers subjected to heat stress.Means ± standard error carry different superscripts within the same raw were significantly different.

Table 4 .
Mean ± standard error of the effect of Amla, Chicory and Leek extracts on serum biochemical parameters of broilers subjected to heat stress.Means ± standard error carry different superscripts within the same raw were significantly different.

Table 5 .
Mean ± standard error of the effect of Amla, Chicory and Leek extracts on liver and kidney function of broilers subjected to heat stress.

Table 6 .
Mean ± standard error of the effect of Amla, Chicory and Leek extracts on antioxidant parameters and oxidative stress enzymes of broilers subjected to heat stress.

Table 7 .
Mean ± standard error of the effect of Amla, Chicory and Leek extracts on total and differential leukocytes count and phagocytic index and activity of broilers subjected to heat stress.Means ± standard error carry different superscripts within the same raw were significantly different.WBCS: white blood cells.