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Effect of green tea extract and vitamin C mix on growth performance and corticosterone concentration in heat-stressed broilers

Nguyen Duy Hoan, Truong Huu Dung, Nguyen Thi Ngan, Phan Thi Hong Phuc, Tu Trung Kien and Phung Duc Hoan

Thai Nguyen University of Agriculture and Forestry,Tan Thinh Ward, Thai Nguyen City 50000, Viet Nam
ndhoan@tnu.edu.vn

Abstract

Heat stress is a major problem in the poultry industry, especially during summer months at tropical countries. Previous studies have reported that green tea extract (GTE) or vitamin C supplementation could palliate the effects of heat stress in broiler chickens. The present study evaluated the effect of dietary single addition of either GTE (10 g/kg) or vitamin C (200 mg/kg) or in combination of GTE and vitamin C (10 g GTE + 200 mg vitamin C per kg) on performance of broilers from 0 to 42 days of age. A total of 576 one day old male birds of Ross 308 chickens were randomly allocated to 96 pens (6 birds/pen) for the purpose of creating 4 groups (24 pens/group). The control group received no additive; the GTE group received 10 gram of GTE/kg; the GTEC group received 10 gram of GTE and 200 mg of vitamin C/kg; the vit-C group received 200 mg of vitamin C/kg of feed). Birds from 4 groups were fed the same diet based on a 3 periods (0 - 10, 11 - 25 and 26 - 42 days). In conclusion, the dietary single addition of either GTE (10 g/kg) or vitamin C (200 mg/kg) or in combination of GTE and vitamin C (10 g GTE + 200 mg vitamin C per kg) provide similar results namely, reduce serum cortocosterone level and mortality, and increase growth performance of broilers reared under heat stress of the same temperature.

Key words: blood serum, nutrition mix, plasma


Introduction

A high environmental temperature is one of the most important factors that causes heat stress among chickens and negatively affects poultry production (Meremikwu et al 2013; Lara and Rostagno 2013). In fact, chickens are confronted with three different ranges of temperature zones—the comfort zone, critical zone and upper critical zone. In the comfort zone (18–25 °C), chickens can maintain their body temperature with minimum effort, while in the critical zone (26–35 °C), maintaining body temperature requires the help of physical heat regulation. Furthermore, in the upper critical zone (higher than 35 °C), chickens cannot dissipate their body’s heat and physiological disorders appear following multi-organ dysfunction, resulting in death (Figure 1). For newly created high-yield broiler breeds, the maximum temperature allowed is 33 °C (Humphrey 2006; Lara and Rostagno 2013).

Figure 1. The temperature zones for chicken welfare (Youssef et al 2015)

Generally, under heat stress condition, chickens attempt to maintain their body temperature within the comfort zone to ensure the function of all vital organs. Thereby, they limit feed intake, walking and standing while increasing resting time, drinking and panting (Suganya et al 2015). When broilers are exposure to heat stress, plasma corticosterone levels increase due to the activation of the hypothalamus - pituitary - adrenal axis (Filho et al 2010; Quinteiro - Filho et al 2012). The biggest impact of heat stress on poultry farms is that may negatively affect feed digestibility, increasing fat storage, reducing yield and meat quality, thereby decreasing the economic efficiency of the farmer. There are many solutions to reduce the effects of heat stress on broiler chickens, in which nutrition factors are of particular interest (Meremikwu et al 2013; Piekarski et al 2015). Previous studies have shown that green tea leaves contain a number of active ingredients, especially epigallocatechin (EGCG), that could help to reduce heat stress in humans and animals. The effect of EGGG in reducing the impact of heat stress is explained that EGCG is a catechin and an ester of epigallocatechin and gallic acid (Farahat et al 2016), which helps to regulate serum metabolites, such as reduced levels of uric acid, cholesterol and triglycerides, activation of creatine kinase, lactate dehydrogenase and aminotransferase (Orhan et al 2013; Himani Vishnoi et al 2018;Alireza Seidavi et al 2019). Supplementing some vitamins such as C, A, E is one of the approaches from a nutritional perspective to prevent the negative effects of heat stress in poultry. Ascorbic acid (vitamin C) is a water-soluble antioxidant compound, which protects cells against oxidative damage and improves immune system function (Abidin et al 2013). Vitamin C is a co-factor of dopamine beta hydroxylase, which participates in the conversion of dopamine to norepinephrine in neural tissues (Harrison and May 2009). Vitamin C has also been shown to regulate the body’s temperature, synthesis of 1.25-dihydroxy vitamin D, and function of the immune system. Emerging literature revealed that adult poultry are capable of synthesizing vitamin C to meet their requirements in normal conditions. However, it has been found that their requirements increase during stress, and several studies have reported the beneficial effects of the dietary supplementation of poultry feed with ascorbic acid (Combs et al 2016). The new features of this study is to determine the effect of a combination of two factors (GTE and vitamin C) on some of the serum parameters related to heat stress and growth performance of broilers reared under heat stress.


Material and methods

Experimental design and feed

Study designed by completely randomized method at HTM farm, Thai Nguyen province, Vietnam. A total of 576 one-day-old male birds of Ross 308 broiler were randomly allocated to 96 similar pens (pen size: 1.0 m x 0.5 m x 0.47 m) with 6 birds /pen for creating of 4 groups (24 pens/ group). The control group received no additive; the GTE group received 10 g of GTE/kg; the GTEC group received 10 g of GTE and 200 mg of vitamin C /kg and the vit-C group received 200 mg of vitamin C/kg of feed. All birds were fed the same diet based on a 3 periods (starter: 0 -10 days, grower: 11 - 25 days and finisher: 26 - 42 days). The formulated diets were based on 3 basic ingredients (maize, wheat and soybean meal, Table 1). The feed was produced in pellet form and provided freely to birds during the study. Green tea extract product was provided by ND Chemical - CAS 84650-60-2; Incoterms: FOB, CFR, CIF, DDP. Polyphenolic basic ingredients of green tea extract (% DM): Epigallocatechin gallate: 7.09, Epicatechin gallate: 3.78, Epigallocatechin: 3.38, Epicatechin: 1.61, Gallocatechin gallate: 1.59; Gallocatechin: 1.55, Catechin: 0.69 – Total: 19.69.

Table 1. Composition and nutrient content of experimental diets

Ingredients, %

Starter
(0-10 days)

Grower
(11–25 days)

Finisher
(26–42 days)

Maize

31.59

18.99

17.32

Wheat

30.08

50.28

54.49

Soy bean meal (47% CP)

26.54

20.67

17.09

Soya oil

4.12

3.43

4.90

Soya protein concentrate (66% CP)

3.8

3.8

3.8

Monocalcium phosphate

1.20

0.60

0.30

Calcium Carbonate

1.22

0.77

0.70

Salt

0.20

0.17

0.17

Sodium bicarbonate

0.22

0.20

0.20

L-Lysine HCl

0.22

0.25

0.25

DL-Methionin

0.24

0.23

0.22

L-Threonine

0.04

0.07

0.08

L-Valine

0.01

0.02

0.01

Enzyme #

0.07

0.07

0.07

Coccidiostat ##

0.05

0.05

0.00

Mineral and vitamin premix###

0.40

0.40

0.40

Feed analysis

ME, kcal

2,850

2,934

3,000

DM (%)

91.56

91.74

91.68

CP (%)

21.02

19.58

18.12

Ether extract (%)

6.56

6.88

7.96

Ash (%)

5.42

4.30

3.89

Calcium (%)

1.01

0.92

0.83

Phosphorus (%)

0.97

0.86

0.78

# Endo-1.3 ß-glucanase; Endo-1.4-ß-xylanase ## For starter period: nicarbin + narasin; For grower and finisher periods: natri monensin. ### Content/kg of diet: 10,000 IU vitamin A; 2,500 IU vitamin D3; 50 IU vitamin E; 2 mg vitamin B1; 6 mg vitamin B2; 40 mg B3; 4 mg vitamin B6; 25 mcg vitamin B12; 2 mg vitamin K3; 1 mg axit folic; 150 mcg d-biotin; 0.25 mg Na2SeO3; 67.7 mg FeSO4 ·7H2O; 1 mg I; 15 mg CuSO4·5H2O; 90 mg MnSO4; 80 mg ZnO

Animal Management

The present study was followed recommendations of the conditions related to animal ethics and welfare described in Section 2, Chapter 5 of the Law on Animal Husbandry Number 32/2018/ QH14 issued by the National Assembly of the Socialist Republic of Vietnam on November 19, 2018. Temperature and humidity were adjusted from the 3rd day of age based on the average annual temperature and humidity during the period from May to June in Vietnam (Table 2). Monitoring of temperature and humidity was carried out continuously with a data logger place in different locations in the chicken house. The amount of water was supplied according to the actual needs of the treatment chickens. Lighting mode was implemented as recommended by Ross broiler handbook (2018). In the first 3 days of lighting 24/24 hours, the following days to implement 16:00 am/ 8 pm.

Table 2. Temperature and humidity at different times of day at the study area

Time (h)

Starter (0-10 days)

Grower (11-25 days)

Finisher (26-42 days)

Temperature
(°C)

Humidity
(%)

Temperature
(°C)

Humidity
(%)

Temperature
(°C)

Humidity
(%)

0

26

75

27

76

26

76

2

26

76

28

76

26

77

4

27

76

28

76

27

76

6

27

75

29

75

28

75

8

28

74

30

75

29

74

10

31

73

32

74

31

73

12

34

72

35

74

34

72

14

36

73

36

75

35

72

16

35

74

35

75

34

72

18

34

75

34

76

33

73

20

30

75

31

76

30

74

22

27

76

28

75

27

75

24

26

75

27

76

26

76

Parameter measure

For determining the mean values of BW, ADG, ADFI and FCR, birds and feed remaining were weighed at the first and the last day of each period. The number and weight of death birds per pen were recorded daily to determine the mortality and used to adjust the FCR for each period. At 4 different time points (8:00 h, 10:00 h, 12:00 h and 14:00 h) of day 21, 6 ml of blood samples at the wing vein from a random bird of all pens were collected (samples were taken from different birds at each time). All blood samples were centrifuged at 3,000 × g at 4 °C for 15 minutes to separate the serum. The Seven EAS device was used to determine the serum pH of all blood samples. Serum samples taken from 12:00 hours onwards were stored deeply refrigerated (-80°C) for analysis of corticosterone and TBARS parameters. Using the IDS immunoassay kit of Boldon, UK to analyze these two criteria, the specific analysis procedure as recommended by the supplier.

Data analysis

The SAS 9.4 software and PROC MIXED of Statistical Analysis System (SAS Institute, Cary, NC, USA) were used to analyse the effect of GTE and vitamin C on growth performance and content of metabolites in blood serum. The Tukey test were used for determining the difference between the mean values between groups. The statistically significant difference was determined when P < 0.05.


Results

For the overall study (0 - 42 days; Table 3), ADG of the GTEC and the vit-C increased compared with that with the control (P < 0.05). The individual supplementation of vitamin C (vit-C group) or a combination supplement with GTE (GTEC group) both increased ADFI compared with that with the control (P < 0.05). FCR of GTEC was significantly reduced compared with that with control (P < 0.05); FCR of GTE and vit-C was mediated between of GTEC and control. The mortality rate of birds of all treatment groups decreased compared with that with the control (P < 0.05). The mortality rate of birds of GTEC group (4.86%) was lower than that of the other group from 2.08 – 4.16% (Figure 2.)

Table 3. BW, ADG, ADFI, FCR and mortality of birds for the whole study (0-42 D)

Group

Final BW
g

ADG
g

ADFI
g

FCR

Mortality
%

Control

2,487.50a

58.14a

95.46a

1.642a

9.02a

GTE

2,538.47ab

59.35a,b

96.74a,b

1.630a,b

7.63 b

GTEC

2,599.23b

60.80b

98.25b

1.616b

4.86c

Vit-C

2,575.42b

60.23b

98.11b

1.629a,b

6.94b

SEM

18

0.48

0.67

0.004

1.08

P value

<0.001

<0.001

0.015

0.016

0.145

a,b,c Values within a column with different letters different p< 0.05)



Figure 2. Mortality of birds for the whole study (0-42 D)

The quantitative results of blood serum pH have shown that the correlation among treatment factors and serum pH has not been determined (Figure 3). However, a comparison at 4 time points showed that blood serum pH at 12:00 h was significantly higher than that at 8:00 h and 10:00 h (P < 0.05). Due to the influence of treatment factors, the serum corticosterone content (ng / mL) of all treatment groups decreased significantly compared with that with the control (P < 0.05; Table 4; Figure 4). However, the concentration of thiobarbituric acid reactive substances (TBARS) was not affected by the treatment factors (Figure 5)

Figure 3. Blood serum pH at 21 days


Table 4. Concentration of blood serum plasma corticosterone and thiobarbituric acid reactive substances (TBARS) at 21 days

Group

Plasma corticosterone
Concentration ( ng/ml)

TBARS
(nmol)

Control

9.84a

96.24

GTE

6.84b

93.02

GTEC

6.76b

92.13

Vit-C

7.12b

94.05

SEM

0.018

0.023

a,b Values within a column with different letters differ at p<0.05



Figure 4. Plasma corticosterone concentration ( ng/ml) at 21 days


Figure 5. Thiobarbituric acid reactive substances concentration (nmol) at 21 days


Discussion

Heat strees is the result of many factors of which ambient temperature is the main one. Heat stress occurs when the ambient temperature is higher than the permissible limit for each type of bird (Humphrey, 2006). Depending on the severity and length of time, heat stress can reduce feed consumption and cause many adverse physiological reactions of the body of birds (Lara and Rostagno 2013; Quinteiro-Filho et al 2012). Heat stress caused the reduced growth rate and the increased mortality (Akbarian et al 2016). Heat stress affects poultry production because it induces oxidative stress, activates the in vivo antioxidant system to remove free radicals and this increased activation requires consumption more energy (Akbarian et al 2016). In addition, the oxidative stress also causes lipid peroxidation and damage protein oxidation, reduce cell and mitochondrial function, and reduce antioxidant vitamin content in blood serum and tissue (Jacob 1995)

There are some ways to reduce the negative influence of heat stress on poultry, including the use of special nutrients, some of which has been tested effectively (Talebi and Khademi 2011; In-Surk Jang et al 2014; Saraeia et al 2016).

The positive effects of green tea have also been reported by Alireza Seidavi et al (2019) that green tea can be used as a growth stimulant in place of antibiotics, it improves food intake, final body weight and nutrient utilization efficiency. On the other hand, EGCG found in green tea extract, is able to prevent the increase of heat shock proteins in the liver through a directly link to the head and acts as a special structural substances (Orhan et al 2013). Additionally, EGCG can limit cell damage and minimize the effects of oxidative stress on poultry (Govinthasamy Prabakar et al 2016).

Dietary supplementation with vitamin C limited and alleviated stress metabolic signs, improved performance, enhanced immunological status and reduced mortality. Previous studies have reported that supplementing with ascorbic acid (vitamin C) increased weight and quality of carcass (Attia et al 2017; Alshelmani et al 2020), reduced blood serum corticosterone concentration in heat-stressed broiler chickens (Mahmoud et al 2004) . According to Saiz Del Barrio et al (2020) adding a combination of electrolytes and vitamin C to the drinking water of heat-stressed broilers improved growth performance. Mahmoud et al (2004) reported that vitamin C was effective in reducing heat shock protein 70 and plasma corticosterone response in broilers ( Gallus gallus domesticus) during heat shock. According to Attia et al (2017) some vitamins, including vitamin C, have a positive effect on heat stress because they have an effect on oxidative stress caused by heat stress. Oxidative stress is understood when the animal is under heat stress, if not controlled, the levels of some reactants such as reactive oxygen, nitrogen and chlorine exceed the available antioxidant capacity in animal cells. This can cause a chain reaction leading to disorders of protein, lipid and nucleic acid metabolism (Akbarian et al 2016). Antioxidant vitamins, including vitamin C, have been shown to protect membrane destruction against ROS damage (Harrison et al 2009).

Abd El - Hack et al (2020) and Alshelmani et al (2020) have demonstrated that green tea extract and vitamin C have the reducing effect on heat stress. On the other hand, previous studies also showed to compare with individual supplements, the supplement combines green tea extract or vitamin C with other nutrients such as minerals, vitamin E, fish oil, corn oil had a better effect on reducing the impact of heat stress on poultry (Imik et al 2012; Afsharmanesh and Sadaghi 2013). The latest finding from this study have shown that group supplemented with a combination of GTE and vitamin C (GTEC group) had a better effect compared with other groups on reducing of negative effects in heat - stressed broiler. This can be explained by the positive resonance of some substances in tea leaves such as catechin, epigallocatechin, epigallocatechin with vitamin C. According to Abd El - Hack et al (2020) adding 0.2 – 1.0% green tea extract to broiler diets reduced feed conversion ratio (FCR) by 8%, abdominal fat by 10 – 20%. The addition of 1% green tea extract to layer hen diets increased egg yield by 5.6%, egg weight by 6.8% and feed conversion ratio by 7.8%. In addition, this study has proven that epigallocatechin gallate was 100 times more effective in neutralizing free radicals than vitamin C, 25 times more effective than vitamin E. Green tea extract has antioxidant and immunostimulating effects for broilers when adding 125 to 500 mg / kg in their diets (El - Deek et al 2012; Farahat et al 2016). The data obtained in this study is similar to that of some other authors such as Yang et al (2003), Huang et al (2013), Keyvan Jelveh et al (2018) thereby demonstrated the bioactive substances contained in GTE have the ability to reduce the negative effects of heat stress on broilers. The literature survey indicated that the optimum response in terms of the growth performance, feed conversion ratio, feed efficiency, survival rate and carcass quality in broilers under heat stress appeared to occur with average supplements of 250 mg/kg vitamin C (Majid Shakeri et al 2020). In present study, adding vitamin C individually (200 mg/ kg of diet) improved growth performance, reduced blood serum corticosterone concentration, reduced feed consumption and mortality. Thus, it can be concluded that adding vitamin C to broiler diets could minimize the negative effects of heat stress on poultry. However, vitamin C supplementation combined with GTE is more effective than individual supplementation. This is also consistent with the research results of Talebi and Khademi (2011) when combining vitamin C with glucose and Ali et al (2010) when combining vitamin C with electrolyte. The addition of GTE and vitamin C to reduce serum corticosterone concentrations has been published by some authors such as Afsharmanesh et al (2013), Saiz del Barrio et al (2020). In this study, levels of GTE and vitamin C were added to the diet based on previous studies, to determine the optimal level of supplementation, more research is needed.


Conclusions


Acknowledgement

The authors are very grateful to the Ministry of Agriculture and Rural Development, Ha Noi, Vietnam for their funding for this project. We would like to thank also the engineers and interns student at the Experimental Center and the Life Science Research Institute, Thai Nguyen Province University of Agriculture and Forestry who participated in this study.


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