Comparative Effect of Zinc Oxide and Silymarin on Growth, Nutrient Utilization and Hematological Parameters of Heat Distressed Broiler

Asad Sultan1, Sahal Ahmad1, Sarzamin Khan1, Rifat Ullah Khan2,*, Naila Chand1, Muhammad Tahir3 and Shakoor Ahmad2

1Department of Poultry Science, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar

2Department of Animal Health, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar

3Department of Animal Nutrition, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar

ABSTRACT

The purpose of the study was to investigate the comparative beneficial effects of dietary supplementation of silymarin (Silybum marianum) and zinc oxide (ZnO) in broilers during the finishing stage exposed to heat stress (HS). Two hundred and seventy broiler chicks were randomly assigned to 6 dietary groups: three dietary groups in thermoneutral temperature (TN) and three dietary groups under high temperature. One group of birds received a basal diet only (control group), second group of birds received a diet supplemented with silymarin at the level of 10g/kg and third group of birds received a diet supplemented with ZnO at the level of 60 mg/kg in both TN and HS birds. It was found that supplementation of ZnO significantly (P<0.05) improved feed intake, dressing percentage, body weight, feed conversion ratio, blood parameters and nutrient digestibility at both levels of thermal zones. The results of silymarin on growth, nutrient digestibility and hematological parameters were comparatively lower (P<0.05) in broilers during TN and HS zones. The current results indicate that the supplementation of feed with silymarin and ZnO improved the growth performance, blood parameters and nutrient digestibility in broiler during heat stress, however, the positive effect of ZnO was better than silymarin.

Article Information

Received 16 November 2017

Revised 19 December 2017

Accepted 31 December 2017

Available online 27 March 2018

Authors’ Contribution

AS, NC and SK designed the study. SA conducted the study. RUK, MT, SA edited the paper.

Key words

Broilers, Digestibility, Heat stress, Silymarin, Zinc oxide.

DOI: http://dx.doi.org/10.17582/journal.pjz/2018.50.2.751.756

* Corresponding author: rifatullahkhhan@gmail.com

0030-9923/2018/0002-0751 $ 9.00/0

Copyright 2018 Zoological Society of Pakistan

Introduction

 

In subtropical countries, high ambient temperature and humidity have harmful effects on production (Chand et al., 2016) and reproduction (Ihsanullah et al., 2017) leading to huge economic losses (Khan et al., 2011a; Zia ur Rehman et al., 2018). As a general rule, animals have known zones of survival and adaptability. For birds, the thermoneutral (TN) zone ranges between 18 to 22°C. Birds are considered heat stressed, when the temperature of their bodies exceeds the upper limit of the TN zone (Khan et al., 2012a; Zia ur Rehman et al., 2017a). Heat stress (HS) adversely affects the growth performance and nutrient digestibility in broiler (Zia ur Rehman et al., 2017b). A number of remedies have been suggested to combat HS, however, the most suitable among them is the dietary supplementation (Khan et al., 2014; Chand et al., 2014; Shakirullah et al., 2017).

The use of antibiotic as feed additive has recently been banned in many countries of the world due to the potential threat of bacterial resistance and antibiotic residues in meat of broiler (Khan et al., 2012b, c; Abudabos et al., 2016; Zia ur Rahman et al., 2017; Alhidary et al., 2017). Therefore, the poultry researchers and nutritionists are looking for alternative to antibiotics, which maintain the growth the birds and do not compromise the health of the consumers (Abudabos et al., 2017a, b; Khan et al., 2012d). Milk thistle (Silybum marianum) is a natural herb and contains various flavonolignans such as silybin, isosilybin, silychristin and silydianin (Surai, 2015). Silymarin has renal, hepatic, anti-carcinogenic and anti-inflammatory effects (Manna et al., 1999). In addition, silymarin and silybin are the two most important flavonoids in milk thistle, which have been reported to stimulate the immune system and scavenge free radical (Thyagarajan et al., 2002; Wilasrusmee et al., 2002). Studies are scarce on the effect of milk thistle in broiler under heat stressed condition. To the best of our knowledge, Silymarin has not been used in the heat stressed broiler. Milk thistle contains some very important compounds such as silybin and flavonolignans, isosilybin, silydianin, dehydrosilybin and silychristin (Alhidary et al., 2017). Silymarin stabilizes and protects the cell membrane against the injury, prevents the deposition of collagen fibre and disrupts free radicals (Surai, 2015).

Zinc (Zn) is a vital element and involved in many biochemical reactions of animal body (Khan et al., 2011b; Rahman et al., 2014). The zinc ion is a cofactor of many enzymes and an element of matallo enzymes that contributes in metabolic processes (Zowczak Drabarczyk et al., 2004; Naz et al., 2016). High ambient temperature can reduce the retention of Zn in poultry birds with increased Zn elimination and alleviates the effects of high temperature in broilers (Sahin et al., 2005). A number of studies have reported the effect of ZnO and silymarin in broilers; however, to the best of our knowledge, their comparative effects have not been reported.

The objective of the present study was to investigate the comparative effect of silymarin and zinc oxide (ZnO) on broiler growth performance, nutrient digestibility and hematology under TN and HS conditions during the finishing phase.

 

Materials and methods

Experimental design and bird husbandry

A total of 270 experimental broiler chicks were divided into two groups and sub-divided into six sub-groups with 3 replicates each. Birds in TN zone were maintained at 35°C and then the temperature was gradually brought and maintained at 23°C. The average temperature and humidity in heat stressed (HS) zone was 38°C and 61%, respectively. A standard diet was prepared as per National Research Council (1994) requirements of the bird (Table I). Zinc oxide and milk thistle powder were added at 60 mg/kg and 10 g/kg of diet, respectively. The experiment lasted for three weeks.

Performance measurement

Offer amount of feed and measure refused feed every day for the determination of feed intake. On weekly basis, the data were analyzed. Bird’s weight was measured on day first of the week and then at the end of the week. On weekly basis body weight gain was determined. The FCR given was recorded on a weekly basis. From each replicate two birds were randomly selected, weighed and slaughtered. The edible and non-edible parts were removed after scalding to find the carcass weight.

Hematology

For hematological study, at the end of the experiment, three ml blood samples per treatment were collected from wing vein in separate ethylene diamine tetra acetate (EDTA) tubes. Red blood cell (RBC), white blood cell (WBC), packed cell volume (PCV) and hemoglobin (Hb) contents were determined with the aid of Cell-DYN 3500 Hematology Analyzer.

Nutrient digestibility and chemical analysis

On day 30, 4 birds per replicate were shifted to metabolic cages for digestibility study. Fecal material was collected for four days and feed intake was determined. Fresh feces were stored in labeled plastic bags and stored at -20°C. Excreta samples were analyzed for nitrogen (Kjeldahl method; AOAC, 1990), CP (N × 6.25), dry matter (drying in oven at 103°C for 8 h), crude fibre (AOAC, 1990), ether extract (AOAC, 2000) and ash (Vogtmann et al., 1975). Metabolizable energy (ME) was determined with the help of bomb calorimeter as described by Sultan et al. (2015).

Statistical analysis

The data were statistically analyzed with the standard procedure (ANOVA) using two factorial designs (2 x 3) in SAS (2004). LSD test was applied according to measure means for significant difference (Steel and Torrie, 1981).

 

Table I.- Composition of basal diets.

Ingredients	(%)
Maize	60.7
Soybean meal	35.6
Vegetable oil	0.5
Limestone	1.4
Dicalcium phosphate	1.2
DL Methionine	0.15
Salt	0.4
Vitamin + trace mineral premix	0.3
Total	100
Calculated nutrient composition
Protein, %	22
Poultry ME, kcal/kg	2960

Each kg of premix consisted: pyridoxine, 1mg; folic acid, 0.4mg; molybdenum, 0.32mg; ethoxyquin, 25mg; choline chloride, 60mg; dI-α-tocopherol acetate; 4mg; iodine, 0.2mg; thiamine, 0.3mg; Ca pantothenate, 3mg; cyanocobalamin, 3µg; biotin, 0.02mg; Mn, 15mg; Zn, 10mg; iron, 4mg; Cu, 1mg; Co, 0.06mg; Se, 0.02mg; cholecalciferol, 0.018mg; trans-retinol, 0.66mg; menadione, 0.4mg; riboflavin, 1.6mg; niacin, 6mg.

 

Results

 

The effect of supplementation of ZnO and milk thistle on the weekly feed intake and weight gain in broiler is given in Table II. Feed intake and weight gain was significantly (P<0.05) high in birds supplemented with ZnO in TN zone. Birds in HS condition showed significantly higher (P<0.05) feed intake and weight gain in response to the supplementation of ZnO.

 

Table II.- Effect of supplementation of zinc oxide and silymarin on feed intake (g), body weight gain (g), feed conversion ratio (FI/BW) and carcass yield (%) of heat stressed broilers.

Tempe rature zone	Treat ments	Feed intake (g)			Body weight gain (g)			FCR (FI/BW)			Carc ass yield (%)
		Day 28	Day 35	Day 42	Day 28	Day 35	Day 42	Day 28	Day 35	Day 42
TN	Control	1523c	2121c	2830c	1010c	1318d	1656c	1.50b	1.60a	1.70b	60.35c
	Silymarin	1588b	2248b	2896b	1086b	1446b	1776b	1.45c	1.55b	1.63d	61.15b
	Zinc oxide	1620a	2326a	3023a	1150a	1533a	1873a	1.40d	1.51c	1.61e	62.34a
HS	Control	1436d	1970d	2720d	920e	1183e	1550d	1.55a	1.66a	1.75a	59.76d
	Silymarin	1485c	2148c	2813c	995d	1351c	1670c	1.49c	1.58c	1.68c	60.15c
	Zinc oxide	1518b	2226b	2923b	1036c	1426b	1780b	1.46d	1.56d	1.64d	61.64b
Pooled SEM		12.01	13.23	14.09	11.09	12.03	13.07	0.07	0.07	0.09	0.95
Temper ature	P value	0.04	0.05	0.04	0.02	0.04	0.04	0.01	0.04	0.03	0.03
Treatment	P value	0.03	0.03	0.02	0.01	0.02	0.03	0.02	0.02	0.02	0.02
Temp × Treatment	P value	0.05	0.06	0.05	0.04	0.05	0.05	0.04	0.05	0.05	0.04

Means within the same column with different superscript are significantly different (P<0.05). TN, thermoneutral; HS, heat stressed.

 

Table III.- Effect of supplementation of zinc oxide and silymarin on blood parameters of heat stressed broilers.

Temperature zone	Treatments	WBC (× 103/µl)	RBC (× 106/µl)	Hb (%)
TN	Control	7.33c	2.53c	9.38d
	Silymarin	8.53b	3.54b	12.37b
	Zinc oxide	9.50a	4.45a	13.50a
HS	Control	6.33d	2.13e	8.12e
	Silymarin	7.46c	2.89d	11.18c
	Zinc oxide	8.43b	3.21b	12.17b
Pooled SEM		0.13	0.19	0.25
Temperature	P value	0.02	0.05	0.04
Treatment	P value	0.01	0.03	0.02
Temp × Treatment	P value	0.04	0.04	0.05

Means within the same column with different superscript are significantly different (P<0.05). TN, thermoneutral; HS, heat stressed.

 

The effect of supplementation of ZnO and milk thistle on the FCR in broiler is given in Table II. The FCR was significantly (P<0.05) high in birds supplemented with ZnO during HS condition compared to the control. Improved FCR was also found in TN condition in ZnO supplemented birds at the end of the experiment.

The carcass yield during TN and HS condition in broiler fed with ZnO and milk thistle is given in Table II. The carcass yield was significantly (P<0.05) high in ZnO supplemented group compared to the control in TN zone. Similarly, ZnO supplemented birds also showed significantly (P<0.05) higher carcass yield during HS condition.

The effect of supplementation of ZnO and milk thistle on the hematological parameters in broiler is given in Table III. The WBC, RBC and Hb were significantly (P<0.05) high in ZnO supplemented birds in TN zone. Similarly, in HS condition, ZnO supplemented birds showed better hematological values compared to the control.

The effect of supplementation of ZnO and milk thistle on the nutrient digestibility in broiler is given in Table IV. ZnO supplementation significantly (P<0.05) increased DM, ash, CP, EE, CF and AME were significantly (P<0.05) high in ZnO supplemented group in TN condition. Similarly, birds during HS, showed better nutrient digestibility in response to ZnO supplementation compared to the control.

 

Table IV.- Effect of supplementation of zinc oxide and silymarin on nutrient digestibility (%) of heat stressed broilers.

Temp. zone	Treatment	DM (%)	Ash (%)	CP (%)	EE (%)	AME (Kcal/kg)
TN	Control	70.18e	30.22e	63.18e	53.25e	2922.74c
	Silymarin	73.16c	31.35b	64.15c	54.44c	3011.89b
	Zinc oxide	74.19a	32.15a	65.08a	55.45a	3113.00a
HS	Control	69.55e	29.22e	62.15e	52.35e	2984.30e
	Silymarin	72.22d	30.26d	63.22d	53.49d	3082.67c
	Zinc oxide	73.25b	31.18c	64.25b	54.50b	3186.11a
Pooled SEM		1.9	0.91	1.8	2.7	13.01
Temp	P value	0.04	0.02.	0.04	0.05	0.01
Treatment	P value	0.03	0.01	0.02	0.03	0.03
Temp × Treatment	P value	0.05	0.04	0.05	0.04	0.04

Means within the same column with different superscript are significantly different (P<0.05). TN, thermoneutral; HS, heat stressed; DM, dry matter; CP, crude protein; EE, ether extract; CF, crude protein; AME, apparent metabolizable energy.

 

Discussion

 

The feed intake of broilers was significantly high during TN as compared to HS zone. Above 32°C is considered a high ambient temperature for birds and the high ambient temperature suppress the feed intake. Zinc oxide and silymarin combat heat stress via its anti-oxidative and free radical scavenging properties. Silymarin supplemented group had higher feed intake as compared to control and ZnO (supplemented) groups in both the zones. Chand et al. (2011) reported that feed intake of broiler chicken fed on Silymarin supplemented diets significantly improved. It may be due to its positive effects on metabolism and digestion. ZnO scavenges free radicals and alleviates stress (Naz et al., 2016).

The feed efficiency and body weight gain per birds were significantly affected by high environmental temperature and treatments assigned. High ambient temperature exerts some hazardous effects on bird’s performance, creating some physiological changes and stresses in body (Chand et al., 2017). Supplementation of silymarin and ZnO attenuate the deleterious heat-induced stress. Supplementation of Zn in diet is necessary for optimal growth, because Zn ion is a cofactor of many enzymes and contributing in metabolism (Zowczak-Drabarczyk et al., 2004). Silymarin promotes digestibility and absorption of protein in animal’s body. All the above reasons might explain, why high weight gain and better FCR were observed in treatment groups compared to the control group. Chand et al. (2011) recoded better performance of birds fed on toxin contaminated feed with silymarin as compared to other birds. Similarly, Tedesco et al. (2004) reported better FCR in birds supplemented with silymarin. Sahin et al. (2005) and Ezzati et al. (2013) reported improved performance of birds in heat stressed condition, when supplement with dietary Zn. The increment in dressed weight is strongly correlated with the improved performance of bird fed on silymarin and ZnO. Increase dressed weight of treated groups is the positive outcome of silymarin and ZnO supplementation in ration. Higher dressing percentage was reported by Chand et al. (2011) and Zahid and Durrani (2007) by feeding milk thistle at different levels in broiler chicken.

In the current study, there is a significant relationship between heat stress and decrease in hematological parameters. Heat stress inhibits production and decreases the life span of RBCs and reduces the Hb level. The WBC showed a significant reduction at high temperature. This might be linked to the atrophy of lymphoid tissue. Also stressed hormones (corticosteroids) have suppressive effects on lymphoid tissue and total leucocytes count. It is pertinent to note that silymarin and ZnO were not much effective in HS zone as compared to the TN condition. At TN zone, the stress was lowered, so beneficial effects of silymarin and ZnO were more pronounced than HS zone. Our results are in agreement with Osman (1996) who reported that the blood parameters revealed a reduction with heat stress.

The digestibility of DM, ASH, CP, EE, CF and Apparent Metabolisable Energy had significant differences. Heat stress reduces glucose and minerals metabolism and produces lesions in gastro intestinal tract (GIT), resulting in low digestibility (Khan et al., 2011). This will lead to an alteration in starch, amino acids, fat, and mineral metabolism, that results in the deposition of abdominal fat and reduction of structural protein. Virden et al. (2007) reported that stress results in significant changes in digestibility of carbohydrates, protein, lipid, and mineral metabolism. It is speculated that supplementation of silymarin and ZnO overcome the heat stress problems in birds, probably due to the reduction in oxidative stress (Chand et al., 2011; Naz et al., 2016).

 

Conclusion

 

Heat stress adversely affected the bird performance, nutrient digestibility and hematology. The supplementation of silymarin and ZnO decreased the negative effects of high stress and improved bird performance. The positive effect of ZnO was better than silymarin in birds both during TN and HS conditions.

 

Statement of conflict of interest

Authors have declared no conflict of interest.

 

References

 

Abudabos, A.M., Alyemni, A.H., Dafalla, Y.M. and Khan, Y.M. 2017a. Effect of organic acid blend and Bacillus subtilis alone or in combination on growth traits, blood biochemical and antioxidant status in broiler exposed to Salmonella typhimurium challenge during the starter phase. J. appl. Anim. Res., 45: 538-542. https://doi.org/10.1080/09712119.2016.1219665

Abudabos, A.M., Alyemni A.H., Dafalla, Y.M. and Khan, R.U., 2017b. The effect of phytogenics on growth traits, blood biochemical and intestinal histology in broiler chickens exposed to Clostridium perfringens challenge. J. appl. Anim. Res., 46: 691-695. https://doi.org/10.1080/09712119.2017.1383258

Abudabos, A.M., Alyemni A.H., Dafallah, Y.M. and Khan, R.U., 2016. The effect of phytogenic feed additives to substitute in-feed antibiotics on growth traits and blood biochemical parameters in broiler chicks challenged with Salmonella typhimurium. Environ. Sci. Poll. Res., 23: 24151-24157. https://doi.org/10.1007/s11356-016-7665-2

Alhidary, I.A., Zia ur Rehman, Khan, R.U. and Tahir, M., 2017. Anti-aflatoxin activities of milk thistle (Silybum marianum) in broiler. World’s Poult. Sci. J., 73: 559-566. https://doi.org/10.1017/S0043933917000514

AOAC, 1990. Official methods of analysis, 15th ed. Association of Official Analytical Chemists, Washington, DC.

AOAC. 2000. Official Methods of Analysis.17th Ed., INTERNATIONAL, Gaithersburg, MD, Official Method 920.39

Sultan, A., Imran ud din, Khan, S., Ullah, R., Khan, H., Khan, N.A. and Khan, R.U., 2015. Effect of yeast derived carbohydrate fraction on growth performance, apparent metabolizable energy, mineral retention and gut histomorphology of broilers during starter phase. Pak. Vet. J., 35: 409-413.

Chand, N., Muhammad, D., Durrani, F., Qureshi, M.S. and Ullah, S.S., 2011. Protective effects of milk thistle (Silybum marianum) against aflatoxin B1 in broiler chicks. Asian-Aust. J. Anim. Sci., 24:1011-1018.

Chand, N., Muhammad, S., Khan, R.U., Alhidary, I.A. and Zia ur Rehman, 2016. Ameliorative effect of synthetic γ-aminobutyric acid (GABA) on performance traits, antioxidant status and immune response in broiler exposed to cyclic heat stress. Environ. Sci. Poll. Res., 23: 23930-23935. https://doi.org/10.1007/s11356-016-7604-2

Chand, N., Naz, S., Khan, A., Khan, S. and Khan, R.U., 2014. Performance traits and immune response of broiler chicks treated with zinc and ascorbic acid supplementation during cyclic heat stress. Int. J. Biometeorol., 58: 2153-2157. https://doi.org/10.1007/s00484-014-0815-7

Chand, N., Naz, S., Maris, H., Khan, R.U., Khan, S. and Qureshi, M.S., 2017. Effect of betaine supplementation on the performance and immune response of heat stressed broilers. Pakistan J. Zool., 49: 1857-1862. https://doi.org/10.17582/journal.pjz/2017.49.5.1857.1862

Ezzati, M.S., Bozorgmehrifard, M., Bijanzad, P., Rasoulinezhad, S., Moomivand, H., Faramarzi, S., Ghaedi, A., Ghabel, H. and Stabraghi, E., 2013. Effects of different levels of zinc supplementation on broilers performance and immunity response to Newcastle disease vaccine. Eur. J. exp. Biol., 3: 497-501.

Ihsanullah, Qureshi, M.S., Suhail, S.M., Akhtar, S. and Khan, R.U., 2017. Postpartum ovarian activities, blood metabolites and milk yield are influenced by changing levels of thermal stress in crossbred dairy cows. Int. J. Biometerol., 61: 1561-1569. https://doi.org/10.1007/s00484-017-1335-z

Khan, R.U., Naz, S., Nikousefat, Z., Tufarelli, V., Javadani, M., Rana, N. and Laudadio, V., 2011a. Effect of vitamin E in heat-stressed poultry. World’s Poult. Sci. J., 67: 469-478. https://doi.org/10.1017/S0043933911000511

Khan, R.U., Nikousefat, Z., Javadani, M., Tufarelli, V. and Laudadio, V., 2011b. Zinc-induced molting: production and physiology. World’s Poult. Sci. J., 67: 497-506. https://doi.org/10.1017/S0043933911000547

Khan, R.U., Naz, S., Nikousefat, Z., Selvaggi, M., Laudadio, V. and Tufarelli, V., 2012a. Effect of ascorbic acid in heat-stressed poultry. World’s Poult. Sci. J., 68: 477-490. https://doi.org/10.1017/S004393391200058X

Khan, R.U., Naz, S., Javadani, M., Nikousefat, Z., Selvaggi, M., Tufarelli, V. and Laudadio, V., 2012b. The use of turmeric (Curcuma longa) in poultry diets. World’s Poult. Sci. J., 68: 97-103. https://doi.org/10.1017/S0043933912000104

Khan, R.U., Naz, S., Nikousefat, Z., Tufarelli, V. and Laudadio, V., 2012c. Thymus vlugaris: alternative to antibiotics in poultry feed. World’s Poult. Sci. J., 68: 401-408. https://doi.org/10.1017/S0043933912000517

Khan, R.U., Naz, S., Tufarelli, V. and Laudadio, V., 2012d. Potential applications of ginger (Zingiber officinale) in poultry diet. World’s Poult. Sci. J., 68: 245-252. https://doi.org/10.1017/S004393391200030X

Khan, R.U., Naz, S. and Dhama, K., 2014. Chromium: Pharmacological applications in heat stressed poultry. Int. J. Pharmacol., 10: 213-317. https://doi.org/10.3923/ijp.2014.213.217

Laudadio, V., Dambrosio, A., Normanno, G., Khan, R.U., Naz, S., Rowghani, E. and Tufarelli, V., 2012. Effect of reducing dietary protein level on performance responses and some microbiological aspects of broiler chickens under summer environmental conditions. Avian Biol. Res., 5: 88-92. https://doi.org/10.3184/175815512X13350180713553

Manna, S.K., Mukhopadhyay, A., Van, N.T. and Aggarwal, B.B., 1999. Silymarin suppresses TNF-induced activation of NF-kappa B, c-jun N-Terminala kinase and apoptosis. J. Immunol., 163: 6800-6809.

National Research Council, 1994. Nutrient requirements of poultry, 9th rev. ed. Natl. Acad. Press, Washington, DC.

Naz, S., Idris, M., Khalique, M.A., Zia-ur-Rahman, Alhidary, I.A., Abdelrahman, M.M., Khan, R.U., Chand, N., Farooq, U. and Ahmad, S., 2016. The activity and use of zinc in poultry diet. World’s Poult. Sci. J., 72: 159-167. https://doi.org/10.1017/S0043933915002755

Osman, A.A., 1996. Effect of heat stress and salts on blood picture of chicken during rearing period. PhD thesis, Faculty of Agriculture, Cairo University.

Rahman, Z., Naz, S., Khan, R.U. and Tahir, M., 2017. An update on the potential application of L-carnitine in poultry. World’s Poult. Sci. J., 73: 823-830. https://doi.org/10.1017/S0043933917000733

Rahman, H., Qureshi, M.S. and Khan, R.U., 2014. Influence of dietary zinc on semen traits and seminal plasma antioxidant enzymes and trace minerals of Beetal bucks. Rep. Domest. Anim., 48: 1004-1007. https://doi.org/10.1111/rda.12422

SAS, 2004. SAS User’s Guide. Statistics Inst., Inc. Cary NC.

Sahin, K., Smith, M.O., Onderci, M., Sahin, M.N., Gursu, M.F. and Kucuk, O., 2005. Supplementation of zinc from organic or inorganic source improves performance and antioxidant status of heat-distressed quail. Poult. Sci., 84: 882-887. https://doi.org/10.1093/ps/84.6.882

Shakirullah, Qureshi, M.S., Akhtar, S. and Khan, R.U., 2017. The effect of vitamin E and selenium on physiological, hormonal and antioxidant status of Damani and Balkhi sheep submitted to heat stress. Appl. Biol. Chem., 60: 585-590. https://doi.org/10.1007/s13765-017-0313-9

Steel, R.G.D. and Torrie, J.H., 1981. Principles and procedures of statistics: A biometrical approach. pp. 625, McGraw-Hill International Book Company, New York.

Surai, P.F., 2015. Silymarin as a natural antioxidant: An overview of the current evidence and perspectives. Antioxidants, 4: 204-247. https://doi.org/10.3390/antiox4010204

Tedesco, D., Domeneghini, C., Sciannimanico, D., Tameni, M., Steidler, S. and Galletti, S., 2004. Efficacy of silymarin-phospholipid complex in reducing the toxicity of aflatoxin B1 in broiler chicks. J. Poult. Sci., 83: 1839-1843. https://doi.org/10.1093/ps/83.11.1839

Thyagarajan, S., Jayaram, S., Gopalakrishnan, V., Hari, R., Jeyakumar, P. and Sripathi, M., 2002. Herbal medicines for liver diseases in India. J. Gastroenterol. Hepatol., 17: S370-S376. https://doi.org/10.1046/j.1440-1746.17.s3.30.x

Virden, W.S., Lilburn, M.S., Thaxton, J.P., Corzo, A., Hoehler, D. and Kidd, M.T. 2007. The effect of corticoster one-induced stress on amino acid digestibility in Ross broilers. Poult. Sci., 86:338-342. https://doi.org/10.1093/ps/86.2.338

Vogtmann H., Pfirter, H.P. and Prabucki, A.L. 1975. A new method of determining metabolisability of energy and digestibility of fatty acids in broiler diets. Br. Poult. Sci., 16:531-534.

Wilasrusmee, C., Kittur, S., Shah, G., Siddiqui, J., Bruch, D., Wilasrusmee, S. and Kittur, D.S. 2002. Immunostimulatory effect of Silybum marianum (milk thistle) extract. Med. Sci. Monit., 8: 439-443.

Zahid, R. and Durrani, F.R., 2007. Biochemical, hematological, immunological and growth promotant role of feed added milk thistle (Silybum marianum) in broiler chicks. M. Sc. (Hons) thesis submitted to NWFP Agric. Univ. Peshawar, Pakistan.

Zia ur Rehman, Chand, N. and Khan, R.U., 2017a. The effect of vitamin E, L-carnitine and ginger on production traits, immune response and antioxidant status in two broiler strains exposed to chronic heat stress. Environ. Sci. Poll. Res., 24: 26851-26857

Zia ur Rehman, Chand, N., Khan, S. and Khan, R.U., 2017b. Evaluating the immune response and antioxidant potential in four broiler strains under chronic high ambient temperature. Pakistan J. Zool., 49: 2087-2091.

Zia ur Rehman, Chand, N. Khan, R.U., Khan, S. and Qureshi, M.S., 2018. An assessment of the growth and profitability potential of meat-type broiler strains under high ambient temperature. Pakistan J. Zool. 50: 429-432.

Zowczak-Drabarczyk, M., Torlinska, T., Iskra, M., Mi elcarz, G., Matylla, G. and Torlinski, L., 2004. Serum zinc concentration in patients colorectal cancer. Trace Elem. Electrolyt., 21: 236-239. https://doi.org/10.5414/TEP21236