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Growth performance, intestinal morphology, and meat quality in relation to alpha-lipoic acid associated with vitamin C and E in broiler chickens under tropical conditions

ABSTRACT

This study was conducted to examine the effect of alpha-lipoic acid with vitamin C and E on growth performance, intestinal morphology, and meat quality in broiler chickens under tropical conditions. A total of 288 one-day-old male ROSS 308 chicks (40±0.1 g) were used in a completely randomized design and allotted to one of six dietary treatments to form sixe replicates per treatment (eight birds per cage). The six dietary treatments were: a corn-soybean meal-based diet (NC; no antimicrobial compounds added) with 8 ppm alpha-lipoic acid (ALA); 150 ppm vitamin C and 75 ppm vitamin E (E-75); E-75 plus ALA (E-75-ALA); 150 ppm vitamin C and 50 ppm vitamin E (E-50) plus ALA (E-50-ALA); and 150 ppm vitamin C and 25 ppm vitamin E (E-25) plus ALA (E-25-ALA). All dietary treatments were continuously provided in liquid form, dissolved in water. Birds were housed in a battery cage (n = 36), and were offered dietary treatments on an ad libitum basis. The ambient temperature was maintained at 32±1 ºC for the first three weeks and reduced gradually to 28 ºC by the end of the experiment (day 35) to induce moderate tropical condition. One bird per pen (n = 6), and another bird per pen (n = 6) were euthanized via cervical dislocation to obtain terminal ileum to measure villus height and crypt depth at day 21, and to harvest breast meat and drumsticks to evaluate meat quality traits at day 35, respectively. Dietary treatment E-75-ALA improved body weight and average daily gain compared with birds fed other dietary treatments from day 1 to day 35. Birds fed dietary treatment E-75-ALA and E-50-ALA had higher villus height than those fed the other dietary treatments at day 21. Dietary treatments E-75-ALA and E-50-ALA reduced thiobarbituric acid reactive substance (TBARS) in drumsticks compared with other dietary treatments, but only treatment E-75-ALA decreased TBARS in breast meat at day 35. Liquid form of antioxidant compounds such as E-75-ALA can improve growth performance, histology of terminal ileum, and meat quality traits in broiler chickens under moderate tropical condition for 35 days.

Key Words:
broilers; heat stress; vitamin E; tropical condition

Introduction

Heat stress is known to be one of the most detrimental factors in overall poultry production (Lara and Rostagno, 2013Lara, L. J. and Rostagno, M. H. 2013. Impact of heat stress on poultry production. Animals 3:356-369. ). Although many studies have clearly elucidated the thermoneutral zone of broilers (18-22 oC; Charles, 2002Charles, D. R. 2002. Responses to the thermal environment. p.1-16. In: Poultry environment problems: a guide to solutions. Charles, D. R. and Walker, A. W., eds. Nottingham University Press, UK. ), it is not easy to maintain that temperature in a specific area or a period or the combination of both. Recent studies have demonstrated that heat stress has affected broiler productivity by reducing feed intake and efficiency, body weight, meat quality, and survivability (Sohail et al., 2012Sohail, M. U.; Hume, M. E.; Byrd, J. A.; Nisbet, D. J.; Ijaz, A.; Sohail, A.; Shabbir M. Z. and Rehman H. 2012. Effect of supplementation of prebiotic mannan-oligosaccharides and probiotic mixture on growth performance of broilers subjected to chronic heat stress. Poultry Science91:2235-2240. ; Lu et al., 2007Lu, Q.; Wen, J. and Zhang, H. 2007. Effect of chronic heat exposure on fat deposition and meat quality in two genetic types of chicken. Poultry Science86:1059-1064. ). Also, it has been shown that heat stress had immunosuppressant effects on birds and resulted in decreasing weights of lymphoid organs, total circulating antibodies, and phagocytic ability of macrophages (Quinteiro-Filho et al., 2012Quinteiro-Filho, W. M.; Rodrigues, M. V.; Ribeiro, A.; Ferraz-de-Paula, V.; Pinheiro, M. L.; Sa, L. R. M.; Ferreira, A. J. P. and Palermo-Neto, J. 2012. Acute heat stress impairs performance parameters and induces mild intestinal enteritis in broiler chickens: Role of acute hypothalamic-pituitary-adrenal axis activation. Journal of Animal Science 90:1986-1994. ). Basically, birds attempt to maintain their thermal homeostasis from heat stress. During this process, the level of reactive oxygen species (ROS) is increased, the body enters a stage of oxidative stress, and heat shock proteins are released in response to stress, providing protection from ROS effects (Droge, 2002Dröge, W. 2002. Free radicals in the physiological control of cell function. Physiological. Reviews 82:47-95. ). Therefore, it can be assumed that antioxidant supplementation to the diet could alleviate impaired productivity of broilers in response to heat stress.

Recently, alpha-lipoic acid (ALA) has been used as a potent antioxidant agent, ranging from therapeutic application to dietary supplementation, widely distributed in foods and readily absorbed from the diet (Packer et al., 1995Packer, L.; Witt, E. H. and Tritschler, H. J. 1995. Alpha-lipoic acid as a biological antioxidant. Free Radical Biology & Medicine19:227-250. ). Reed (1957Reed, L. J. 1957. The chemistry and function of lipoic acid. Advances in Enzymology 18:319-347. ) demonstrated the basic functions of ALA, including its antioxidant properties for the first time in leaves. Later, Packer et al. (1995)Packer, L.; Witt, E. H. and Tritschler, H. J. 1995. Alpha-lipoic acid as a biological antioxidant. Free Radical Biology & Medicine19:227-250. reported that ALA acted on dehydrogenase complexes as a co-factor. Also, ALA and its reduced form, dihydrolipoic acid (DHLA), had a function as antioxidants in free-radical quenching, metal chelation, antioxidant recycling, and gene expressing (Packer et al., 1995). Likewise, ALA had the scavenging ability directly against the hydroxyl radical, the hypochlorous radical, and singlet oxygen, but not against the superoxide radical and the peroxyl radical (Suzuki et al., 1991Suzuki, Y. J.; Tsuchiya, M. and Packer L. 1991. Thioctic acid and dihydrolipoic acid are novel antioxidants which interact with reactive oxygen species. Free Radical Research15:255-263. ; Scott et al., 1994Scott, B. C.; Aruoma, O. I.; Evans, P. J.; O'Neill, C.; Van der Vliet, A.; Cross, C. E.; Tritschler, H. and Halliwell, B. 1994. Lipoic and dihydrolipoic acids as antioxidants. A critical evaluation. Free Radical Research 20:119-133.; Kagan et al., 1992Kagan, V. E.; Shvedova, A.;; Serbinova, E. Khan, S.; Swanson, C.; Powell, R. and Packer, L. 1992. Dihydrolipoic acid: A universal antioxidant both in the membrane and in the aqueous phase - reduction of peroxyl, ascorbyl and chromanoxyl radicals. Biochemical Pharmacology 44:1637-1649. ; Stevens et al., 1974Stevens, B.; Perez, S. R. and Small, R. D. 1974. The photoperoxidation of unsaturated organic molecules: IX. Lipoic acid inhibition of rubrene autoperoxidation. Photochemistry and Photobiology 19:315-316. ). However, DHLA, known to rapidly convert into ALA, is capable of quenching the oxidants that cannot be scavenged by ALA (Kagan et al., 1992Kagan, V. E.; Shvedova, A.;; Serbinova, E. Khan, S.; Swanson, C.; Powell, R. and Packer, L. 1992. Dihydrolipoic acid: A universal antioxidant both in the membrane and in the aqueous phase - reduction of peroxyl, ascorbyl and chromanoxyl radicals. Biochemical Pharmacology 44:1637-1649. ). In addition, ALA has shown antioxidant activity by chelating iron, copper, and other transition metals (Packer et al., 1995Packer, L.; Witt, E. H. and Tritschler, H. J. 1995. Alpha-lipoic acid as a biological antioxidant. Free Radical Biology & Medicine19:227-250. ). Particularly, DHLA interacted with other antioxidants by regenerating cellular antioxidants, such as vitamin E, vitamin C, and glutathione from their radicals or inactive form (Biewenga et al., 1997Biewenga, G. P.; Haenen, G. R. and Bast, A. 1997. The pharmacology of the antioxidant lipoic acid. General Pharmacology: The Vascular System 29:315-331. ). Consequently, it appears that DHLA is involved in indirect antioxidative activity.

Numerous studies have been conducted to evaluate the antioxidative effects of ALA on livestock. Bai et al. (2012Bai, X. M.; Ma, Q. G.; Zhao, L. H.; Xi, L. and Ji, C. 2012. Effects of alpha-lipoic acid supplementation on antioxidative ability and performance of sows and nursing piglets. Journal of Animal Physiology and Animal Nutrition 96:955-961) determined that ALA supplementation to the diet of sows during late-gestation and lactation improved the activity of antioxidant enzymes in the serum and improved the performance of sows and their nursing piglets. In broiler chickens, dietary ALA inhibited body fat deposition, decreased muscle glycolysis at early postmortem, and improved the water holding capacity, indicating that ALA supplementation could prevent the occurrence of PSE (pale, soft, exudative) meat (El-Senousey et al., 2013El-Senousey, H. K.; Fouad, A. M.; Yao, J. H.; Zhang, Z. G. and Shen, Q. W. 2013. Dietary alpha lipoic acid improves body composition, meat quality and decreases collagen content in muscle of broiler chickens. Asian-Australasian Journal of Animal Science 26:394-400. ). Lipid peroxidation level, superoxide dismutase, and catalase enzyme activities and glutathione amounts in Japanese quail under heat stress conditions were ameliorated with the addition of ALA to the diet (Halıcı et al., 2012Halıcı, M.; Imik, H.; Koç, M. and Gümüş, R. 2012. Effects of α-lipoic acid, vitamins E and C upon the heat stress in Japanese quails. Journal of Animal Physiology and Animal Nutrition96:408-415. ). However, few results have been reported regarding the synergic antioxidative effects of dietary ALA together with vitamins C and E in broilers (Sahin et al., 2003Sahin, K.; Sahin, N. and Kucuk, O. 2003. Effects of chromium, and ascorbic acid supplementation on growth, carcass traits, serum metabolites, and antioxidant status of broiler chickens reared at a high ambient temperature (32 C). Nutrition Research 23:225-238.).

The objective of this study was, therefore, to evaluate the effects of antioxidants (ALA, vit. E, and vit. C) on growth performance, ileal morphology, and meat quality in broilers under chronic heat stress conditions. This study was also conducted with the purpose of elucidating the optimum levels of supplemented antioxidants in liquid form to the water supplied to broilers reared at a high ambient temperature.

Material and Methods

The practices and procedures for this experiment were reviewed and approved by the Chungnam National University Animal Ethics Committee (CNU-00521).

A 5-week experiment was conducted with 288 one-day-old male ROSS 308 broilers (40.0±0.1 g). Broilers were individually weighed and randomly assigned to one of six dietary treatments; each treatment had six replicates with eight birds per pen. All birds were raised in stainless steel battery cages of identical size (86 cm width × 57 cm length × 35 cm height) and provided with continuous lighting (24 h). The initial ambient temperature of the room was maintained at 32±1 oC for the first three weeks and decreased gradually to 28 oC, and 60 to 65% humidity was maintained using a room temperature control system by the end of the experiment (day 35). The basal corn-soybean meal diets were formulated to meet or slightly exceed the nutritional requirements of broilers during the starter (days 1-25) and grower (days 26-35) phases, according to NRC (1994) recommendations for broiler chickens (Table 1). The broilers were allowed ad libitum access to feed and fresh water. The six experimental treatments (i.e., liquid form) were given to broilers during the entire experimental period as follows: a corn-soybean meal-based diet (NC; no antimicrobial compounds added) with 8 ppm ALA; 150 ppm vitamin C and 75 ppm vitamin E (E-75); E-75 plus 8 ppm ALA (E-75-ALA); 150 ppm vitamin C and 50 ppm vitamin E (E-50) plus 8 ppm ALA (E-50-ALA); and 150 ppm vitamin C and 25 ppm vitamin E (E-25) plus 8 ppm ALA (E-25-ALA). Birds were monitored for mortality two times a day throughout the experiment.

Table 1
Composition of the experimental diets

Feed intake (FI) and body weight (BW) were recorded weekly per cage, and feed conversion ratio (FCR) was calculated as FI divided by body weight gain (BWG) every week, for a 5-week period. At day 21, one bird per pen was selected to obtain ileum tissue to measure villus height and crypt depth. The birds were euthanized via cervical dislocation after a 12 h fast. Fragments of approximately 5 cm in length were obtained from the ileum, between Meckel's diverticulum and the anterior portion of the ileocecal junction. The excised fragments were immersed in a phosphate-buffered formalin solution. Two portions per sample were cut perpendicular to the longitudinal axis of the intestine and embedded in paraffin wax. Transverse sections were cut (3~5 µm). In the morphometric study, images were captured using a light microscope and a system that analyzes computerized images (Bio-Rad Microscience, UK). The height of 10 well oriented villi and their associated crypts were measured from each replicate per segment. The mean was obtained from these values. At the end of the experiment, one bird per pen was selected randomly. All birds fasted for 12 h were euthanized by exsanguination for carcass data. The carcasses were eviscerated manually and then immediately stored at −20 oC for subsequent analysis.

After the carcasses were thawed at room temperature, each breast and leg meat sample was obtained from each carcass, and then meat quality associated with antioxidative activity was analyzed.

The pH values of leg meat and breast meat were assessed. Briefly, 10 g of sample were homogenized for 30 s using a stomacher (400 Lab blender, Seward, England) in 100 mL distilled water; afterwards, the pH of the sample was measured using a pH meter (WTW pH 720, Germany).

To determine the cooking loss, each sample was cut into 2.5 cm thick slices that were then packaged in polyethylene bags and placed in an 80 oC water bath for 30 min. The samples were then removed and cooled at room temperature for 30 min, after which the cooking loss values were calculated based on the difference in the weight of the meat before and after cooking (Barbanti and Pasquini, 2005Barbanti, D. and Pasquini, M. 2005. Influence of cooking conditions on cooking loss and tenderness of raw and marinated chicken breast meat. LWT-Food Science and Technology 38:895-901. ).

The thiobarbituric acid (TBA) values were measured according to the modified extraction method described by Witte et al. (1970Witte, V. C.; Krause, G. F. and Bailey, M. E. 1970. A new extraction method for determining 2- thiobarbituric acid values of pork and beef during storage. Journal of Food Science 35:582-585. ). Briefly, 10 g of each sample, 15 mL of cold 10% perchloric acid, and 25 mL distilled water were added to this sample. After homogenizing the mixture at 10,000 rpm for 10 s in a homogenizer (AM-Series, Kaisha Ltd., Japan), the homogenate was filtered using qualitative filter paper no. 2. After adding and completely mixing 5 mL of the filtrate solution and 5 mL of 0.02 M TBA solution, the solution was allowed to stand for 16 h in a cool and dark place. The absorbance was measured at 529 nm suing a spectrophotometer (DU-650, Beckman, USA). 1,1,3,3,-Tetra 1,1,3,3,-Tetraethoxypropane (Sigma-Aldrich, St. Louis, MO, USA) was used as standard for TBA assay. Thiobarbituric acid values were expressed as milligrams of malonaldehyde (MA) per kilogram of sample (mg MA/kg), and the standard curve equation used was y = 0.1975x + 0.0011 (r = 0.999), in which y = absorbance for a given x, the TBA value.

The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity of leg and breast meats was determined according to the modified method described by Blois (1958Blois, M. S. 1958. Antioxidant determination by the use of a stable free radical. Nature 181:1199-1200.). Briefly, 1 mL of lemon balm ethanol extract and 1 mL of 0.2 mM DPPH were added to a test tube and mixed for 30 min at 37 °C, and the absorbance of the mixture was measured at 517 nm using a UV-spectrophotometer (Shimadzu UV-1601PC, Japan). At the same time, antioxidant activity was measured by the same method using ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), which is a natural antioxidant, as well as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), which are synthetic antioxidants, as a positive control group. The DPPH radical scavenging activity of each sample was calculated using the following formula: (absorbance of sample addition group/absorbance of the control group) × 100. Ascorbic acid, BHA, and BHT were used at 0.5 mg/mL. Sample concentration providing 50% inhibition (IC50) was calculated from the graph of inhibition percentage against sample concentration.

Data were analyzed univariately in a normal-linear model using the GLM procedure of SPSS (version 21.0, SPSS Inc., Chicago, Illinois, USA). The pen was the experimental unit for the growth performance (i.e., average daily gain [ADG], average daily feed intake [ADFI], and feed conversion ratio [FCR]). Initial BW was included in the model as a covariate for analyses of growth performance data. The individual chicken was considered the experimental unit for intestinal morphology and meat quality traits. Statistical significance was accepted at P<0.05. Pair-wise comparisons between means were made when appropriate using Fisher's protected LSD analysis when a significant treatment effect was observed.

Results

The results indicated the effects of antioxidant supplementation on broiler performance under moderate heat stress (Table 2). No significant difference appeared (P>0.05) in ADFI, FCR, or mortality among dietary treatments. However, birds fed E-75-ALA had higher (P<0.05) ADG than birds fed NC for 35 days (Table 2).

Table 2
Effect of dietary antioxidant on growth performance and mortality rate of broiler chickens from 1 to 35 days of age

Birds fed E-75-ALA and E-50-ALA had improved (P<0.05) villus height on day 21. Nonetheless, birds fed E-75-ALA had lower (P<0.05) crypt depth compared with negative control. Furthermore, birds fed E-75-ALA had higher (P<0.05) villus height:crypt depth ratio compared with NC, PC, and E-75 (Table 3).

Table 3
Effect of dietary antioxidant on villus height and crypt depth of broiler chickens on day 21

No difference was found (P>0.05) in the pH values of leg or breast meat among the dietary treatments (Table 4). Birds fed E-75-ALA had higher (P<0.05) breast and leg meat cooking loss compared with NC (Table 4). Birds fed ALA combination had higher (P<0.05) DPPH radical scavenging activity of leg and breast meat among the dietary treatments. Similarly, birds fed E-75-ALA had lower (P<0.05) thiobarbituric acid reactive substances (TBARS) value in leg and breast meat compared with the negative control.

Table 4
Effect of dietary antioxidant on meat quality traits of broiler chickens on day 35

Discussion

In chicken rearing, heat stress has caused economic loss and concurrent welfare issues. The best way to avoid heat stress is maintaining the right ambient temperature, but it is costly to install a cooling system and keep that ambient temperature in cages, particularly in hot areas or in the summer season. Thus, many studies have focused on manipulating the diet to reduce the physiological and physical damages of broilers from heat stress. In the present study, antioxidants (vit. E, vit. C, and ALA) were added to drinking water, and the combination of antioxidants positively affected growth performance, intestinal morphology, and meat quality under moderate heat stress, suggesting that supplementation of dietary antioxidants could ameliorate the raising and productivity of broiler chickens. Sigel et al. (1995Sigel, H. S. 1995. Gordon memorial lecture. Stress, strains and resistance. British Poultry Science36:3-22. ) reported that growth performance was decreased in birds when the ambient temperature rise was beyond the thermoneutral zone (i.e., over 32 ºC). In the present study, the birds fed a diet without any antioxidants showed decreased final BW and ADG compared with those fed a diet supplemented with vit. C, vit. E (75 ppm), and ALA (Table 2). This result was in agreement with previous reports that indicated decreased BW from heat stress, but that were compensated for by the addition of antioxidants such as ascorbic acid (Imik et al., 2012Imik, H.; Atasever, M. A.; Urcar, S.; Ozlu, H.; Gumus, R. and Atasever, M. 2012. Meat quality of heat stress exposed broilers and effect of protein and vitamin E. British Poultry Science53:689-698. ; Sahin et al., 2003Sahin, K.; Sahin, N. and Kucuk, O. 2003. Effects of chromium, and ascorbic acid supplementation on growth, carcass traits, serum metabolites, and antioxidant status of broiler chickens reared at a high ambient temperature (32 C). Nutrition Research 23:225-238.). Our results suggested that antioxidants may enhance digestibility, resulting in increasing ADG and BW, although no significant difference was observed in ADFI (Table 2). Indeed, Wallis and Balnave (1984Wallis, I. R. and Balnave, D. 1984. The influence of environmental temperature, age and sex on the digestibility of amino acids in growing broiler chickens. British Poultry Science25:401-407. ) reported that high environmental temperatures had detrimental effects on broilers by decreasing their digestibility of amino acids. Larbier et al. (1993Larbier, M.; Chagneau, A. M. and Geraert, P. A. 1993. Influence of ambient temperature on true digestibility of protein and amino acids of rapeseed and soybean meals in broilers. Poultry Science72:289-295.) found that high temperature condition decreased the true digestibility of protein, which might deter the activity of protein digestive enzymes (trypsin, chymotrypsin, and amylase) under heat stress. These negative influences on digestibility were moderated through the supplementation of the antioxidant vitamins. Sahin and Kucuk (2001)Sahin, K. and Kucuk, O. 2001. Effects of vitamin C and vitamin E on performance, digestion of nutrients and carcass characteristics of Japanese quails reared under chronic heat stress (34 C). Journal of Animal Physiology and Animal Nutrition85:335-341. demonstrated that the combination of 200 mg vit. C and 250 mg vit. E improved nutrient digestibility of dry matter, organic matter, crude protein, and ether extract in Japanese quails, which was also confirmed in broilers exposed to heat stress (Sahin et al., 2003Sahin, K.; Sahin, N. and Kucuk, O. 2003. Effects of chromium, and ascorbic acid supplementation on growth, carcass traits, serum metabolites, and antioxidant status of broiler chickens reared at a high ambient temperature (32 C). Nutrition Research 23:225-238.). Nevertheless, we were not in the position to determine digestibility in the present study.

The present study could anticipate that supplementation of antioxidants increased the digestive capacity under heat stress, although there was no effect of antioxidants on feed intake (Tables 2 and 3). It is known that the gastrointestinal tract is sensitive to stressors (Suzuki, 1983Suzuki, K. 1983. The hand and environment (5):--Physiological changes of digital functions in two age groups studied by three stress tests under three environmental conditions. Nihon Seikeigeka Gakkai Zasshi 57:79-89. ; Burkholder et al., 2008Burkholder, K. M.; Thompson, K. L.; Einstein, M. E.; Applegate, T. J. and Patterson, J. A. 2008. Influence of stressors on normal intestinal microbiota, intestinal morphology, and susceptibility to Salmonella enteritidis colonization in broilers. Poultry Science87:1734-1741. ). When broilers were exposed to feed withdrawal and heat stress, ileal morphology became aberrant, leading to increased attachment of Salmonella Enteritidis (Burkholder et al., 2008Burkholder, K. M.; Thompson, K. L.; Einstein, M. E.; Applegate, T. J. and Patterson, J. A. 2008. Influence of stressors on normal intestinal microbiota, intestinal morphology, and susceptibility to Salmonella enteritidis colonization in broilers. Poultry Science87:1734-1741. ). Also, diminished feed intake due to heat stress affected the height of villi in broilers (Tarachai and Yamauchi, 2000Tarachai, P. and Yamauchi, K. 2000. Effects of luminal nutrient absorption, intraluminal physical stimulation, and intravenous parenteral alimentation on the recovery responses of duodenal villus morphology following feed withdrawal in chickens. Poultry Science79:1578-1585. ). It is well known that the villus height:crypt depth ratio is the gut health index (Pluske, 1997Pluske, J. R.; Hampson D. J. and Williams, I. H. 1997. Factors influencing the structure and function of the small intestine in the weaned pig: a review. Livestock Production Science 51:215-236. ). When the villus height:crypt depth ratio was low, the intestinal environment became more favorable to nutrition absorption than vice versa (Pluske, 1997Pluske, J. R.; Hampson D. J. and Williams, I. H. 1997. Factors influencing the structure and function of the small intestine in the weaned pig: a review. Livestock Production Science 51:215-236. ). Caspary (1992Caspary, W. F. 1992. Physiology and pathophysiology of intestinal absorption. The American Journal of Clinical Nutrition 55:299S-308S. ) reported that increasing the villus height is considered a means to broaden the surface area, thereby improving the absorption of available nutrients. Thus, shortening villi can take a toll on the nutrient absorption by reducing the surface area. Proliferation of stem cells is present at the base of the crypt, which differentiates mostly from enterocytes. The enterocytes migrate up the villus and are extruded from the villus tip into the lumen (Imondi and Bird, 1966Imondi, A. R. and Bird, F. H. 1966. The turnover of intestinal epithelium in the chick. Poultry Science45:142-147. ). In the course of the process, the enterocytes become mature and functional in terms of nutrient absorption and mucin secretion (Wright and Alison, 1984Wright, N. A. and Alison, M. 1984. The biology of epithelial cell populations. Oxford University Press, New York. ). In this regard, poor nutrient absorption may occur due to the decrease in villus height and increase in crypt depth. Similarly, Fan et al. (1997Fan, Y. K.; Croom, J.; Christensen, V. L.; Black, B. L.; Bird, A. R.; Daniel, L. R.; McBride, B. W. and Eisen, E. J. 1997. Jejunal glucose uptake and oxygen consumption in turkey poults selected for rapid growth. Poultry Science76:1738-1745. ) reported that the villus height:crypt depth ratio was closely associated with increased epithelial cell turnover. Also, Samanya and Yamauchi (2002)Samaya, M. and Yamauchi, K. E. 2002. Histological alterations of intestinal villi in chickens fed dried Bacillus subtilis var. natto. Comparative Biochemestry and Physiology Part: A Molecular & Integrative Physiology 133:95-104. observed that longer villi were correlated with activated cell mitosis in chickens.

The morphometric analysis results in the present study showed that the supplementation of antioxidants increased villus height but decreased crypt depth in broilers reared under chronic heat stress. The birds supplemented with 150 ppm vit. C, 75 ppm or 50 ppm vit. E, and 8 ppm ALA had significantly higher villus height than negative control, whereas no significant difference occurred between positive and negative control groups (Table 3). Similar results were obtained for crypt depth and villus height:crypt depth ratio, except that supplementation of 150 ppm vit. C, 50 ppm vit. E, and 8 ppm ALA also provided a significantly higher villus height (Table 3). Based on these results, it seems that supplementation of 8 ppm ALA cannot ameliorate impaired intestinal morphology per se. In a sense, antioxidant activities of ALA can be expressed when it is supplemented together with vit. E and vit. C. According to Packer et al. (1995Packer, L.; Witt, E. H. and Tritschler, H. J. 1995. Alpha-lipoic acid as a biological antioxidant. Free Radical Biology & Medicine19:227-250. ), it appears that ALA lacks biochemical capacity for scavenging the superoxide radical, hydrogen peroxide, and the peroxyl radical. Although ALA was ineffective against some oxidants, it has been widely known that ALA effectively interacts with other antioxidants by increasing the antioxidant activity of one another. For example, ALA and vit. C continuously recycled vit. E, which was predominantly used to protect membranes from lipid peroxidation as the major chain breaking antioxidant (Sies et al., 1994Sies, H. 1994. Strategies of antioxidant defense. p.101-107. In: EJB Reviews 1993. Springer, Berlin Heidelberg.; Packer, 1992Packer, L. 1992. New horizons in antioxidant research: Action of the thioctic acid/dihydrolipoic acid couple in biological systems. p.35-44. In: Thioctsaure. 2nd International Thictic Acid Workshop. Universimed Verlag GmbH, Frankfurt.). Alpha-lipoic acid is also capable of recycling vit. E by regenerating vit. C (Biewenga et al., 1997Biewenga, G. P.; Haenen, G. R. and Bast, A. 1997. The pharmacology of the antioxidant lipoic acid. General Pharmacology: The Vascular System 29:315-331. ). In addition, microsomal lipid peroxidation was inhibited by the reduced form of ALA, which is a dihydrolipoic acid in the presence of vit. E (Scholich et al., 1989Scholich, H.; Murphy, M. E. and Sies, H. 1989. Antioxidant activity of dihydrolipoate against microsomal lipid peroxidation and its dependence on α-tocopherol. Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism 1001:256-261. ). Alpha-lipoic acid inclusion in vivo increases the level of ubiquinol, which is the substance known to recycle vit. E under oxidative stress circumstances (Götz et al., 1994Götz, M. E.; Künig, G.; Riederer, P. and Youdim, M. B. 1994. Oxidative stress: free radical production in neural degeneration. Pharmacology & Therapeutics 63:37-122. ; Kagan et al., 1990Kagan, V. E.; Serbinova, E. and Packer, L. 1990. Antioxidant effects of ubiquinones in microsomes and mitochondria are mediated by tocopherol recycling. Biochemical and Biophysical Research Communications 169:851-857. ). A study showed that 500 mg vit. E/kg did not have any effects on villus height, crypt depth, and villus height:crypt depth ratio (Murakami et al., 2007Murakami, A. E.; Sakamoto, M. I.; Natali, M. R. M.; Souza, L. M. G. and Franco, J. R. G. 2007. Supplementation of glutamine and vitamin E on the morphometry of the intestinal mucosa in broiler chickens. Poultry Science86:488-495.). Thus, vit. E may not act solely on the intestinal mucosa effectively. Turan and Mahmood (2007Turan, A. and Mahmood, A. 2007. The profile of antioxidant systems and lipid peroxidation across the crypt-villus axis in rat intestine. Digestive Diseases and Science 52:1840-1844. ) suggested that the liberation of enterocytes from the villus tip cells due to apoptosis generated the large amount of free radicals in villus tip cells (Turan and Mahmood, 2007Turan, A. and Mahmood, A. 2007. The profile of antioxidant systems and lipid peroxidation across the crypt-villus axis in rat intestine. Digestive Diseases and Science 52:1840-1844. ). In the present study, combination of antioxidants (vit. E, vit. C, and ALA) may effectively scavenge the generated free radicals caused by heat stress, consequently resulting in improved ileal morphology.

The detrimental influences in broilers by heat stress were found not only in their growth performance, but also meat quality. Heat stress induced alterations of muscle metabolism and membrane integrity, which could be associated with the meat quality (Lu et al., 2007Lu, Q.; Wen, J. and Zhang, H. 2007. Effect of chronic heat exposure on fat deposition and meat quality in two genetic types of chicken. Poultry Science86:1059-1064. ; Zhang et al., 2012Zhang, Z. Y.; Jia, G. Q.; Zuo, J. J.; Zhang, Y.; Lei, J.; Ren, L. and Feng, D. Y. 2012. Effects of constant and cyclic heat stress on muscle metabolism and meat quality of broiler breast fillet and thigh meat. Poultry Science91:2931-2937.; Sanderock et al., 2001). The pH value of the meat is known to be an important physical factor in the postmortem stage. Because it indicates meat color, cooking loss, and meat sensory quality (Ahn and Maurer, 1990Ahn, D. U. and Maurer, A. J. 1990. Poultry meat color: kinds of heme pigments and concentrations of the ligands. Poultry Science 69:157-165. ; Guignot et al., 1994Guignot, F.; Touraille, C.; Ouali, A.; Renerre, M. and Monin, G. 1994. Relationships between post-mortem pH changes and some traits of sensory quality in veal. Meat Science 37:315-325. ). In this study, the pH value of both leg and breast meat was numerically lower in dietary vit. C, vit. E (75 ppm), and ALA than negative control (Table 4). We assume that antioxidants might have a positive effect on the pH value in breast and drumstick meat during storage. The 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activity is a simple method to evaluate antioxidative activity (Yamaguchi, 1998Yamaguchi, T.; Takamura, H.; Matoba, T. and Terao, J. 1998. HPLC method for evaluation of the free radical-scavenging activity of foods by using 1, 1-diphenyl-2-picrylhydrazyl. Bioscience, Biotechnology, and Biochemistry 62:1201-1204. ; Arshad et al., 2011Arshad, M. S.; Anjum, F. M.; Asghar, A.; Khan, M. I.; Yasin, M.; Shahid, M. and El-Ghorab, A. H. 2011. Lipid stability and antioxidant profile of microsomal fraction of broiler meat enriched with α-lipoic acid and α-tocopherol acetate. Journal of Agricultural Food Chemistry 59:7346-7352.). In the present study, the diets including ALA showed significantly higher DPPH free radical scavenging activity than negative control (Table 4). These results suggest that breast and leg meat from the broilers that drank liquid antioxidants might have electron donors to neutralize free radicals, which is in agreement with results of a previous study (Jung et al., 2010Jung, S.; Choe, J. H.; Kim, B.; Yun, H.; Kruk, Z. A. and Jo, C. 2010. Effect of dietary mixture of gallic acid and linoleic acid on antioxidative potential and quality of breast meat from broilers. Meat Science86:520-526. ). Many studies reported that chickens reared at a high temperatures had increased TBARS in plasma, organs, and carcass (Lin et al., 2000Lin, H.; Du, R. and Zhang, Z. Y. 2000. Peroxide status in tissues of heat-stressed broilers. Asian-Australasian Journal of Animal Science13:1373-1376. ; Mahmoud and Edens, 2003Mahmoud, K. Z. and Edens, F. W. 2003. Influence of selenium sources on age-related and mild heat stress-related changes of blood and liver glutathione redox cycle in broiler chickens (Gallus domesticus). Comparative Biochemistry and Physiology - Part B: Biochemistry & Molecular Biology 136:921-934.; Gao et al., 2010Gao, J.; Lin, H.; Wang, X. J.; Song, Z. G. and Jiao, H. C. 2010. Vitamin E supplementation alleviates the oxidative stress induced by dexamethasone treatment and improves meat quality in broiler chickens. Poultry Science89:318-327.). The supplementation of antioxidants prevented lipid oxidation in chicken meat that decreased TBARS value, which is a consequence of oxidant stress (Jung et al., 2010Jung, S.; Choe, J. H.; Kim, B.; Yun, H.; Kruk, Z. A. and Jo, C. 2010. Effect of dietary mixture of gallic acid and linoleic acid on antioxidative potential and quality of breast meat from broilers. Meat Science86:520-526. ; Cortinas et al., 2005Cortinas, L.; Barroeta, A.; Villaverde, C.; Galobart, J.; Guardiola, F. and Baucells, M. D. 2005. Influence of the dietary polyunsaturation level on chicken meat quality: Lipid oxidation. Poultry Science84:48-55.). The findings in the present study showed that both leg and breast meat samples from the broilers that drank the water supplemented with vit. C, vit. E (75 ppm), and ALA had lower TBARS values than the negative control group, suggesting that vit. E might improve lipid stability and reduce lipid oxidation in chicken meat (Cortinas et al., 2005Cortinas, L.; Barroeta, A.; Villaverde, C.; Galobart, J.; Guardiola, F. and Baucells, M. D. 2005. Influence of the dietary polyunsaturation level on chicken meat quality: Lipid oxidation. Poultry Science84:48-55.). Gao et al. (2010)Gao, J.; Lin, H.; Wang, X. J.; Song, Z. G. and Jiao, H. C. 2010. Vitamin E supplementation alleviates the oxidative stress induced by dexamethasone treatment and improves meat quality in broiler chickens. Poultry Science89:318-327. demonstrated that the high level of vit. E was effective for the performance of broilers by reducing oxidative stress.

Conclusions

Broilers that drink water supplemented with 150 ppm vitamin C, 75 ppm vitamin E, and 8 ppm alpha-lipoic acid reared under high ambient temperature have positive results in growth performance, ileal morphometry, and meat quality. Also, the optimal vitamin E level of 75 ppm when in combination with vitamin C at 150 ppm and alpha-lipoic acid at 8 ppm plays important roles in antioxidant activity, ameliorating heat stress. Birds under heat stress consume more water than food. Consequently, broiler chickens are expected to become less susceptible to heat stress by drinking water supplemented with antioxidants.

Acknowledgments

Financial support from Biogenoci Co. Ltd. is greatly appreciated. This paper was financially supported by the research fund of Chungnam National University. The authors thank Mr. Samiru Sudharaka Wickramasuriya and Mr. Cheol Woo Lee for the technical assistance.

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Publication Dates

  • Publication in this collection
    Mar 2016

History

  • Received
    24 Aug 2015
  • Accepted
    03 Dec 2015
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