Various environmental stressors such as high ambient temperature and relative humidity influence the performance of broilers by reducing feed intake, feed conversion efficiency and hypophyseal-adrenocortical axis that in turn stimulates corticosterone which retarded growth (Dong et al., 2007). Reactive oxygen species (ROS) are generated at cellular level during normal bodily functions; however, high ambient temperature has been shown to increase the free radicals and other ROS production in body fluids and tissues. Their accumulation due to over-production or a decreased antioxidant defense leads to damage of biological macromolecules and disruption of normal cell metabolism (Spurlock and Savage, 1993).
Dietary modifications are among the most preferred and practical ways to alleviate the effect of high environmental temperature in poultry. Furthermore, antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) play a vital role in protecting cellular damage from the harmful effects of ROS (Meister and Anderson, 1983). High ambient temperature depletes such antioxidants and induces oxidative stress. In addition to oxidative stress, marked elevation of temperature increases blood glucose and cholesterol concentrations (Altan et al., 2010). Non-enzymatic antioxidants such as vitamin C (Sahin et al., 2004; Sahin et al., 2003) and herbs containing different anti-stressor activity such as Emblica officinalis have been used to protect tissues from superoxide radicals and enhance cell survival by stimulating anti-oxidative enzymatic systems. Amla (Emblica officinalis) or Indian gooseberry has been traditionally used for many chronic conditions including diabetes in ayurvedic medicine. It is rich in vitamin C (Sanjyal and Sapkota, 2011). Vitamin C has ability to sequester the singlet oxygen radical, stabilize the hydroxyl radical and regenerate reduced vitamin E back to the active state and to control reactive oxidant species formed during exercise (Kaminski and Boal, 1992). The active tannoid principles of E. officinalis are important hypolipidaemic agents that directly act upon the sympatho-adrenal axis and lower the synthesis of corticosterone (Sairam et al., 2003). The hypolipidaemic effect of E. officinalis has been attributed to its potential in reducing lipid peroxidation. Impairment of immunological function in heat stress, such as T and B lymphocyte activity, has also been attributed to the effects of lipid peroxidation or oxidative damage in cell membranes (Pardue and Thaxton, 1986). Therefore, in the present experiment fruit powder of this plant was evaluated in coloured broilers during extreme summer months in the process of screening effective anti-heat stressors.
MATERIALS AND METHODS
All the experiments were carried out as per the code of practice approved by the Institute of Animal Ethics Committee at Central Avian Research Institute, Izatnagar, UP-24122, India (Permission no.: 452/01/ ab/CPCSEA).
Performance of colored broiler (CARI-DHANRAJA) chickens (n= 112, day-old) fed diets with or without addition of amla (Emblica officinalis) fruit powder during extreme summer (May-June, 38° C to 43° C) was assessed in open house rearing system. Fresh fruits were collected from the natural habitat away from the areal pollution, identified, authenticated and then were washed with distilled water. The fruits were then air dried under shed, powdered in an electrical grinder and stored in air tight container at room temperature for use. Four dietary treatments were prepared by adding different levels of dried fruit powder (0, 0.1, 0.2 and 0.3% of diet) in practical broiler starting (0-3 wk) and finishing (3-6 wk) diets (Table 1). Each diet was offered to 4 replicated groups of 7 birds each. The experimental birds were reared group wise in randomly allotted cabins of the electrically heated battery brooders with the provision of wire-mesh floor, feeder and waterer, located in the well ventilated room; with 24 hours light and uniform management following approved welfare practices. Weighed amount of each test diet used during starting (0-3 wk) and finishing (3-6 wk) period was offered daily in quadruplicate lot of 7 chicks each to ensure ad libitum feeding, but with care to avoid spillage and wastage. The fresh and wholesome water was always made available in suitable troughs to all the birds during the study period. Feed consumption was recorded on weekly basis while the body weight gain (BWG) was recorded on 21st (starting) and 42nd (finishing) days of age then feed conversion ratio (FCR, Feed: Gain) was calculated.
Sampling and analysis
Haematological and biochemical indices
These parameters were performed at the end of experiment (6th week age) in eight birds per treatment group. Packed cell volume (PCV,%) in whole blood was determined by capillary microhaematocrit method by centrifugation at 10000 rpm for 15 min. Haemoglobin concentration (g/dl) in the whole blood was estimated by cyanomethemoglobin method (Vankampen and Zinglstra, 1961). Blood smears were prepared from fresh blood was stained by Geimsa stain (1:9 Dilution for 45 min) for differential leucocyte count (DLC). Reduced glutathione (GSH) level in serum was estimated using the method (Lin Hu et al., 1988) with some modifications. Lipid peroxide level in serum was measured by determining the malondialdehyde (MDA) production using thiobarbituric acid (TBA) with slight modifications (Beuge and Aust 1978; Suleiman et al., 1996). Oxidative stress factors (LPO and GSH were multiplied and then divided by PCV) were then calculated.
Humoral and Cell mediated Immunity
The humoral immune response (haemagglutinationHA titre to sheep red blood corpuscles-SRBC) of broiler chicks fed diet with or without addition of Emblica fruit powder were analyzed (Siegal and Gross, 1980; Vander Zijpp, 1983). The cell mediated immune (CMI) response (foot web index to Phytohaemagglutinin, lectin from Phaseolus VulgarisPHAP) was studied on 21st day (Corrier and Deloach, 1990). For immune study, a total of 32 chicks (8 chicks/ treatment) were selected each for cell mediated and humoral immune-response. The weight of lymphoid organs (bursa of Fabricius and spleen) was taken at the end of experiment and expressed as per cent of live weight.
Heat shock protein 70 expression
Six experimental birds [three from control (0% amla) and three from dietary treatment (0.3% amla)] were sacrificed at the end of the 6th wk. The liver and spleen tissues were collected aseptically and preserved for estimation of HSP 70 expression. Total RNA was isolated from the tissues (Liver and spleen) following established TRIzol method. The purity of RNA checked before the preparation of first-strand cDNA. Prepared cDNA stored frozen at -20° C and was used for the HSP 70 expression studies. Expression of HSP 70 was quantified by using specific primer pairs for genes of interest (GOI) in Real-Time PCR. GAPDH used as a reference gene. Oligo-nucleotide sequence of gene primers has been given in table 2.
The data pertaining to various parameters were analyzed statistically by the methods of (Snedecor and Cochran, 1983). The significant mean differences were attributed according to Duncan’s multiple range test (DMRT) (Duncan, 1955). The mRNA expression levels (expression ratio) of genes of interest were analyzed by REST 2009 software. The difference in mean values was considered as significant at the level of 95% (P < 0.05).
The overall body weight gain due to dietary (Table 3) addition of Emblica fruit powder during 0-3, 3-6 and 0-6 wk of age remained statistically unchanged (1239 to 1289 g). No mortality was observed during experimental period at any treatment. Feed intake was significantly (P<0.001) reduced in starter (846 vs 730, 730, 757 g/bird), finisher (1799 vs 1676, 1506, 1716g/bird) and overall growth phase (2627 vs 2406, 2236, 2474 g/bird) in the broilers fed with diet containing 0, 0.1, 0.2 and 0.3% Emblica fruit powder respectively. The feed conversion ratio (kg feed per kg weight gain) during 0-3 wk, 3-6 wk and 0-6 wk phase (1.93, 1.78, 2.01 vs 2.07) were better (P<0.001) in diet with 0.2% Emblica fruit powder compared to other dietary treatments including control. Carcass traits viz. shrinkage-loss, blood loss and relative weight of heart remained comparable but feather loss, giblet, liver, gizzard, eviscerated yield and dressed yield differed among various treatments (Table 4). The yields of wing and thigh remained similar but that of breast (P<0.01), drum stick (P<0.001) and neck (P<0.01) increased with reduction of back yield (P<0.001) on dietary addition of Emblica fruit powder. The humoral immune response against sheep red blood cells, relative weights of bursa and spleen (Table 5) remained comparable, but cell mediated immune response improved (P<0.01) on dietary addition of Emblica fruit powder at any level owing to the adaptogenic and immunomodulatory potential of the polyherbal formulation and vitamin C.
The haemoglobin level, lipid peroxidase activities, oxidative stress factor and H: L ratio (Table 6) remained comparable but lower levels of reduced glutathione (P<0.01) was estimated in broiler chickens fed diets with Emblica fruit powder being lowest at 0.1% level. The mRNA expression of HSP70 in liver and bursa remain comparable to control group (Table 7). However, the mRNA expression level of HSP-70 in spleen was significantly downregulated (P<0.001) in dietary supplemented amla at 0.3% level (in comparison to control group) by a mean factor of 0.506 (Table 7 and Fig. 1). Expression of HSP70 is tissue and age specific.
Reports on Amla incorporation in broiler diet were scarce hence discussions were made with vitamin C as when required. Emblica officinalis belongs to Citrus group, which is a rich source of ascorbic acid (Vitamin C) (Jain and Khurdiya, 2002). Findings of the present study were in line with earlier reports (Tuleun and Njoku, 2000; Pena et al., 2008; Patil et al., 2014) who also reported no beneficial effect of ascorbic acid/ amla supplementation on weight gain in broilers as an anti-stress agent during high environmental temperature. In contrary reports (Njoku, 1986; Vathana, 2002) were shown an increased weight gain in broilers fed ascorbic acid supplemented diet. The incorporation of Emblica at 0.4 and 0.8% resulted in higher body weight gain in broilers than the respective un-supplemented group (Patel et al., 2016). Similar positive observations in broilers were also reported by (Patil et al., 2014; Kumar et al., 2012; Kumar et al., 2013). The higher body weights observed in E. officinalis supplemented groups may be attributed to anabolic and antioxidant effect of ascorbic acid, gallic acid and tannic acids present in E. officinalis (Mcdowell, 1989). The insignificant effect of Emblica in the current study might be due to lesser level of incorporation compared to the earlier levels. The reduction in broiler feed intake fed with Emblica was also observed (Tuleun and Njoku, 2000; Lin et al., 2003). While, no effect of amla or ascorbic acid supplementation on feed consumption was also reported (Kumar et al., 2013; Abdel Raheem and Ghaffar, 2004). In contrary, significantly higher feed consumption was also recorded (Patil et al., 2014; Jadhav, 2005). The results of this experiment are in agreement with available reports (Vathana, 2002; Njoku and Nwazota, 1989), who reported improved feed conversion with ascorbic acid during high temperature. The incorporation of E. officinalis in broiler diet tends to positively alter the yield composition of the broiler carcass. However, the dietary supplementation of ascorbic acid did not cause any significant differences among the analyzed treatments on carcass and cut-up part yields of 33-day-old birds (Pena et al., 2008).
Apart from CMI response to PHA-P, the humoral immunity and immune organs weights were remained unaffected by E. officinalis feeding. The present findings was in line with earlier works in broilers (Lin et al., 2003; Abdel Raheem and Ghaffar, 2004) who also reported that improved immune function with dietary addition of ascorbic acid during heat stress. The ascorbic acid can improve immunity of bird during heat stress as compared to the birds exposed to heat stress without supplementation (Farooqi et al., 2003). Ascorbic acid also takes part in the synthesis of leukocytes especially phagocytes and neutrophils which play a part in the defense system of the chickens (Null, 2011).
The hemoglobin (g%), heterophil: lymphocyte ratio and lipid peroxidase enzyme activity (n mol/ ml) were remained unaffected by different levels of amla feeding. The observations of the present study were similar to previous studies where no change in H: L ratio due to dietary addition of ascorbic acid was reported (Campo and Davila, 2002). In the contrary of present findings, increased haemoglobin concentration in broiler (Njoku and Nwazota, 1989) reported with dietary addition of ascorbic acid. A decrease in lipid peroxidation was recorded when dietary supplementation of antioxidant herbs e.g. active tannoid principles of E. officinalis (Bhattacharya et al., 1999). The practical poultry production imposes multiple and concurrent stressor effects and reported that heterophil: lymphocyte ratios increased linearly and additively as a function of the imposition of single or multiple stressors (McFarlane et al., 1989a; McFarlane et al., 1989b). Ascorbic acid supplementation showed positive effects on the reduction of heterophil/lymphocyte ratio in birds exposed to heat stress (33°C) and supplemented with 150 and 300 ppm ascorbic acid in the diet (McKee and Harrison, 1995). Feeding of broilers with 0.5% of amla in combination with 0.5% graph seed powder reduced the plasma cortisol levels in broiler breeders (Priya et al., 2010).
High ambient temperatures can potentially induce patho-physiological alterations spatially in gastrointestinal tract during heat stress. Heat stress lead to expression of a small group of highly conserved polypeptides, known as heat shock proteins (HSP) in different tissues, by prokaryotic and eukaryotic cells is clearly observed (Leandro et al., 2004). HSP are thought to play a role in cellular protection under high ambient temperature, with a proposed relationship between the development of thermo-tolerance and HSP synthesis, especially HSP70 (Lindquist and Craig 1988). Exposure of chicken to high temperature increased the induction of HSP70 in brain (Zulkifli et al., 2009), liver (Mahmoud and Edens, 2003), lungs and heart (Mahmoud and Edens, 2005) and in quail ovary and brain (Sahin et al., 2009). The present findings got support from earlier work, where supplementation of ascorbic acid leads to decreased HSP70 expression compare to control non-supplemented group under heat stressed environments (Mahmoud et al., 2003). The mechanism of this beneficial effect is not yet clear, but it is believed that supplementation of amla enhances potentially release of bioactive substances that could prevent oxidative damage and ultimately lowers expression of HSP. Also, dietary ascorbic acid supplementation reduced the circulating levels of corticosterone as well as serum cholesterol in the heat-stressed chickens this could result in a decrease in the HSP70 expression (Pardue and Thaxton 1986; Wang and Edens, 1993; Mahmoud et al., 2004).
Finally, it is concluded that Amla (Emblica officinalis) fruit powder 0.2% in diets of coloured broiler chickens (0-42 d of age) was beneficial to improve feed conversion efficiency, cell-mediated immune response while 0.3% was beneficial to improve HSP70 expression during extreme summer.
The author A. B. Mandal expresses his gratitude to the Indian Council of Agriculture Research, New Delhi for providing all facilities and funding. The authors are grateful to the Director, ICAR-Central Avian Research Institute, Izatnagar for providing support to carry out this work under the Institute project.
CONFLICT OF INTEREST
The authors do not have any conflict of interest.
This article was originally published in Journal of Animal Research: v.7 n.2, p. 233-241. April 2017. DOI: 10.5958/2277-940X.2017.00035.3.