Effect of vitamin E supplementation on production and heat shock protein 70 in broiler chickens during hot-humid summer

Chicken meat and egg production has been on the increase in all continents with the highest increases in Asia and South America (Mandal, 2010). High temperatures coupled with high humidity impose severe stress on birds and can cause huge economic loss. Such loss is due to reduced performance, immune suppression, and an increase in respiratory disease and mortality (Mandal, 2010). Exposure of chickens to high temperature also increases the induction of heat shock protein 70 (HSP70) in different tissues including the brain, liver and lungs (Sahin et al., 2009). Considerable attention has been paid to the role of nutritional manipulation in minimizing the effects of heat stress. To reduce heat stress losses antioxidant, vitamins and minerals are commonly enriched in the diets of birds reared under conditions likely to result in heat stress (Sahin and Kucuk, 2003). Antioxidant nutrient supplementation, especially Vitamins E inclusion in diet is effective in heat stressed broilers (Felipe et al., 2015). Since poultry cannot synthesize Vitamin E and its concentration is reduced under heat stress conditions, Vitamin E requirements must be met from dietary sources. The present study was designed to evaluate Vitamin E as anti-heat-stressor under high temperature and humidity conditions.

120 broiler chicks were reared in cages on a standard broiler starter diet (214.4 g/kg CP and 2839 kcal ME/kg) up to 14 days of age and thereafter finisher diet (197.5g/kg CP and 2891 kcal ME/kg) up to 42 days on test diets with or without vitamin E. Dietary treatment groups were three in number viz., T1 (control diet), T2 (vitamin E @ 150mg/kg) and T3 (vitamin E @ 250mg/kg). Each treatment comprised of five replicate of eight birds each. Experiment was carried out during the hot-humid summer (August-September, 26.0±0.12 to 34.2±0.37°C, RH %: 77.0±0.90-86.1±0.61).

Birds and food issues and returns were recorded weekly to 42 days of age and weight gains, food intakes and feed efficiency were calculated over relevant intervals. Mortalities were recorded as they occurred. To assess the HSP70 expression, jejunal samples were collected at d 28 and 42. Total cellular RNA was isolated using RNAgents® Total RNA Isolation System (Promega, Madison, USA), purity and quantity were assessed by measuring the optical density of each sample at 260 versus 280 nm in nanodrop. Total RNA sample (2 μg) was reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (MBI Fermentas, Hanover, USA) according to the manufacturer’s instructions. The qPCR assays were evaluated by the generation of a standard curve. Calibration curves for each gene were done on an iQ5 cycler (Bio-Rad Laboratories, Hercules, USA) with five 10-fold serial dilutions (in triplicates) and were calculated by the Bio-Rad Optical System Software (Version 2.1, 2010) with the analysis mode “PCR base line substracted”. Expression of HSP70 was studied in real time PCR (SYBR® Green Qiagen GmbH, Hilden, Germany) using the designed primers (Table 1). PCR cycling conditions included initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30s, annealing for 30s and extension at 72°C for 45s. The mRNA expression levels (expression ratio) of HSP70 gene were analyzed by REST 2009 software.

Weekly BWG was significantly higher in the vitamin E supplemented treatments between 3 and 4 weeks of age than in the unsupplemented controls; subsequently, there were no significant differences between treatments. The feed intake during different weekly periods except 21st and 35th day period remained comparable. Vitamin E supplementation at both 150 and 250 mg/kg improved feed efficiency each week from 21 to 42 days of age. In agreement with our findings, Sahin et al. (2001) concluded that supplemental vitamin E in broilers reared under heat stress conditions, significantly increased feed intake, body weight gain and helped to improve the overall performance of birds. Supplemental Vitamin E resulted in increased feed and water consumption.

Moreover, vitamin E is involved in reducing free radical formation and thus providing welfare besides less secretion of corticosterone, a catabolic hormone. Figures 1 and 2 indicate the relative fold expressions of HSP70, at 28 and 42 days, respectively, of the two vitamin E treatments compared with the control. Data were calculated according to Pfffal et al. (2002) and showed significant decrease in expression of the HSP70 gene in the vitamin E treatments. At 28th day post-hatch Vitamin E supplementation at 150 mg/kg as well as 250 mg/kg level bring significant (P< 0.01) down regulation in HSP70 gene. Significant (P< 0.05) down regulation in HSP 70 gene is also observed at 42nd day of age. Kapkin et al. (2013) concluded that heat stress reduces the secretion of HSP-70 in testes of broilers, when Vitamin E is administered. Similar to present study, Ushakova et al. (1996) showed that dietary supplements of Vitamin E can modify gene expression induced by heat shock in vivo and have a protective role against oxidative stress by enhancing the level of endogenous antioxidants and down regulating HSP70 gene expressions in mice. Vitamin C and vitamin E are primary antioxidants in biological systems and break the chain of lipid Peroxidation in cell membranes. Several studies have investigated the relationship between HSP expression and vitamin C (Mahmoud and Edens, 2003).

It was concluded that supplementation of Vitamin E at 150mg/kg under heat stress conditions will reduce stress through impact on heat shock proteins, leading to improvements in both growth performance and welfare of birds.