Chronic heat stress (HS) is a great concern in all types of poultry especially broiler chickens. Reductions of feed intake (Teeter ., 1985) and lower growth rate as well as reduction in feed efficiency (Geraert ., 1996) have been reported in poultry under HS condition (Ramnath., 2008). Birds attempt to compensate for their reduced ability to dissipate heat during heat distress by increasing their physiological processes (such as, elevated body temperature, panting and respiratory alkalosis, etc.) (Teeter and Belay., 1996). Further studies have demonstrated that the heat stress(HS)responses in poultry are excited mainly by the activation of hypothalamic–pituitary–adrenal axis and of the orthosympathic nervous system, and changed metabolic status elicited by decreased plasma levels of T3and T4 (Bowen .,1984), Insulin and increased plasma corticosterone (CS) Concentration (Donkoh., 1989; Sahin ., 2003).
Sinurat. (1987) reported that the efficiency of feed utilization and feed intake in broilers was reduced under heat stress and supplementation of antioxidants improved the feed efficiency of broilers. High ambient temperature has been shown to increase the free radicals and other ROS in the body fluids and tissues. Although, the low level of ROS are essential for many biochemical processes but its accumulation due to over-production or a decreased antioxidant defense, leads to damage of biological macromolecules and disruption of normal cell metabolism and physiology (Spurlock and Savage., 1993). Mujahid. (2005) showed that superoxide production by the skeletal muscle mitochondria of meat type chickens is significantly enhanced by heat stress. Lipid peroxides induce a diminution of the production performance of chicks. (Shim., 2006). Maintenance of normal cell functions in the presence of oxygen largely depends on the efficiency of tissue protection against free radicals mediated oxidative stress body has its own defense mechanisms that protects cell against cellular oxidants and prevent their accumulation (Tainiguchi ., 1992).
Heat loss in broilers is limited by feathering and the absence of sweat glands (Teeter., 1985). When the temperature and relative humidity exceed the comfort level of a bird, it loses the ability to efficiently dissipate heat. The protective enzymatic systems of superoxide dismutase (SOD), catalase (CAT), and GSH recycling enzymes could reduce the amount of free radicals and repair the free radical damage of tissues. SOD is the most important enzyme among these enzymes which catalyzes the dismutation of O2 to H2O2. Hence, the changes of this enzyme activity may have an important effect on the free radicals damage under heat stress. Furthermore, antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) play a vital role in protecting cellular damage from 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 in increases blood glucose and cholesterol concentrations is also evident (Altan ., 2000).
L-glutamine is the most prevalent amino acid in the bloodstream, accounting for 30 to 35 percent of the plasma amino acid nitrogen and the free amino acid pool in the body (Souba., 1993). Because glutamine contains two ammonia groups, one from its precursor, glutamate, and the other from free ammonia in the bloodstream, one of glutamine’s roles is to act as a “nitrogen shuttle,” which helps protect the body from high levels of ammonia in the blood (Helton ., 1990). It is well demonstrated that Gln appears to be a conditionally essential amino acid nutrient during stress (e.g. weaning, injury, illness, oxidation and endotoxin) (Helton ., 1990; Shizuka ., 1990; Alverdy ., 1992; Wu ., 1995., 1996). Glutamine also plays a role in eliminating free radicals because it acts as a precursor for the antioxidant glutathione synthesis (Wu., 1998). The role of glutamine in glutathione synthesis suggests that the availability of this nutrient may have a profound effect on the regulation of the cellular redox status.
Glutamine supplementation in poultry and swine diets has been reported to beneficial in several aspects (Yi ., 2001; Kitt ., 2002). Exogenous glutamine had the effect of antioxidant protection for rats with the implanted tumor (Kaufmann ., 2007) and mice with dystrophic muscles (Mok ., 2008). As a precursor of glutathione (GSH), glutamine also showed its anti-inflammatory and anticancer effects by up-regulating the gut GSH metabolism in the post-sepsis or murine models of asthma (Kaufmann ., 2008; Singleton ., 2008). The objective of the current study was to assess the effect dietary glutamine supplementation on performance, some blood antioxidant indices of broiler chickens under continuous heat stress condition.
Materials and methods
A total of two hundred one-day-old broiler chicks (Ross 308) were obtained from a commercial hatchery. Chickens were weighed and allotted into 5 groups randomly. Each treatment comprised 5 replicate pens with 10 birds each. All chickens had ad lib access to water and feed and the diets were available as mash from. Diets were based on corn-wheat- soybean meal and formulated to meet the chicken’s recommended levels of Ross requirements (Ross Company). Diets had similar nutritive value (Table 1). The chickens were fed the same starter (from day 1 to day 21of age) and grower (from day 22 to day 42 of age) diets throughout the whole experiment but received different levels of 0.0, 0.25, 0.5, and 1 percent glutamine. Chicks were raised at 32±1 °C for inducing heat stress from day one to the end of the experimental period (day 42 of age). Birds were exposed to 23h and 1h darkness during the experiment.
Body weight gains (BWG), feed intake (FI) and feed conversion ratio (FCR) were determined for the starter, grower and whole the experimental periods. At the end of the experiment (week 6), five birds per treatment were randomly selected and slaughtered. At slaughter, two series of blood samples were collected in anticoagulant tubes (EDTA). The blood samples immediately transferred to laboratory, then one series of blood samples was centrifuged at 5000 rpm for 5 min and their plasma separated and stored at -20 °C along with other series of blood samples for the later analyses. Plasma TAC was determined using Randox total antioxidant status test kit (Randox Laboratories Ltd, UK), blood SOD activity by Ransod spectrophotometric kit (Ransod, Randox Laboratories Ltd. UK), blood GPX activity by Ransel spectrophotometric kit (Ransel, Randox Laboratories Ltd. UK) and plasma MDA concentration by MDA reaction with thiobarbituric acid followed by extraction with butanol (Kolahi ., 2011). The data were analyzed based on a completely randomized design using the GLM procedure of SAS (SAS Institute, 2003). Duncan’s multiple range tests was used to separate the means when treatment means were significant (P≤ 0.05). The experimental protocols were reviewed and approved by the Animal Care Committee of the Urmia University.
Table 1. Ingredient composition and chemical analysis of the experimental diets (g kg – 1, as fresh matter)
Results and discussion
The effect of dietary Glutamine on BWG, FI and FCR during weeks 1 o 6 of age and whole the experimental periods is shown in Tables 2. BWG of MG birds were higher than those of ZG and LG birds during the whole period (P<0.05). FI and FCR was not affected by dietary Gln supplementation during the weeks 1 to 6 of age or entire the period (P>0.05).
Table 2. Average feed intake (FI, g d –1), Body weight gain (BWG, g d –1) and Feed conversion ratio (FCR) of broiler chickens fed 0.0 (ZG), 0.25 (LG), 0.5 (MG) and 1 % Glutamine supplementation (HG) from day one to day 42 of age under heat stress conditions
At week 6 of age, none of the blood TAC and MDA contents or SOD and GPX activities were affected by dietary treatments (P>0.05) (Table 3). In a recent study, dietary supplementation of 0.5 percent Glutamine (Gln) improved BWG of broiler chickens during the entire period. The positive effect of Gln has been well established in broiler chickens. For example, 0.5 percent Gln in diet increased body weight gain, feed consumption and decreased feed conversion ratio of broilers under heat stress 22 to 42 day. Moreover dietary including 0.5 percent Gln along with 100 mg/kg GABA cussed improved body weight gain, feed consumption and feed conversion ratio of broilers under heat stress (34±2) 22 to 42 day during(Dai.,2011). The dietary 0.5 percent Gln + 0.5 percent GABA cussed improve performance broilers under heat at 22 to 42 day experimental during (Dia ., 2011). Soltan (2009) and fasina .(2010) had demonstrated that dietary supplementation of Gln increased growth performance of broiler in thermo neutrality. it is demonstrated that Gln appears to be conditionally essential amino acid nutrition during stress (e.g . weaning, injury, illness, oxidation and endotoxin) as exogenous Gln can be a potential candidate in improving intestinal morphology , digestive function (Helton .,1990; Shizuka ., 1990; Alverdy .,1992; Wu ., 1995,1996) and intestinal enzymes (Protease , Lipase, Lactase, Sucrase) (WU .,1996; lin and Xiao 2006; Dia .,2009b).
Table 3. Blood activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX) enzymes and total antioxidant capacity (TAC) of 0.0 (ZG), 0.25 (LG), 0.5 (MG) and 1% Glutamine supplementation (HG) fed broiler chickens at day 42 of age under continues heat stress condition.
The finding concurs with sousadias and smith (1995) and Yi .(2005) who reported better body weight in broilers fed putrescine and L- glutamine at 1% level. Priya (2010) indicated on the role of levels 0.5 and 1% Gln in chicks intestinal enzyme activity was scarce, the result was correlated with rats. The disaccharides activity was significantly (p<0.05) higher in L-glutamine fed during 2 week of age. The lipase activity was not influenced both at 2 and 3 weeks of age(Priya.,2010). Increased villi height has been proposed to increase performance by improving nutrient absorption (Coates ., 1954; Izat ., 1989). The increase in villi height that was observed might indicate that the birds fed diets supplemented with 1% Gln might have had greater nutrient absorption and utilization because increases in villi height result in more surface area for nutrient utilization.
The increase in surface area might also explain the significantly heavier intestinal relative weights (P < 0.05) and improved weight gains that were observed due to Gln supplementation. Even through the birds fed diets supplemented with 4% Gln had increased villi height and actually had the longest villi in comparison with the controls or the 1% Gln, they had the lowest growth performance. This may be due an imbalance in amino acids in the 4% Gln diet, or it could also suggest that in fact, increased villi height does not necessarily lead to increased nutrient utilization and then performance (Bartell,2005). The broilers supplemented with the commercial mixture of L-glutamine and L-glutamate (0.5, 1.0 and 1.5%) exhibited a better performance compared to unsupplemented groups (Avellaneda.,2008).
Stressed states e.g., trauma and sepsis, the intestinal uptake of Gln from the blood is significantly increased (Nemoto ., 1996). The addition of a mixed diet with aspartic acid and glutamine significantly improved the broiler weight gain (De- lian.,2009). Supplementing a standard corn-soybean meal diet with 1% glutamine (old source) for 21 or 40 days (entire experimental period) improved broiler growth performance in all four studies and feed efficiency in the last three studies. Supplementing the diet with 1% glutamine increased villi height, intestinal relative weights, thymus and spleen relative weights, IgA, IgG, IgM and IFN-? concentrations, and anti-SRBC titers in broiler chicks (Bartell, 2006).
In addition to enhanced survival these studies have shown that glutamine improved nitrogen balance, diminished the sepsis induced decrease in muscle glutamine concentration, and decreased muscle protein breakdown (Ardawi, 1991), increased plasma glutamine concentration (Inoue ., 1993), increased intestinal function andor integrity (Inoue ., 1993; Naka, 1996), and enhanced muscle protein synthesis (Ardawi, 1991; Naka, 1996). Liu. (2002) suggested that the jejunal atrophy was prevented by 1.0% glutamine supplementation during the first week post-weaning piglets. Yu. (2002) also suggested that a combination of 1.0% of glutamine and 1000 ppm of nucleotide in diet could improve feed intake and intestinal villus height.
The reasons being that glutamine facilitated the survival and proliferation of intestinal mucosal cells and that glutathione synthesis from glutamine maintains the mucosal integrity and defenses. Another explanation could be the glutamine-dependent protein expression of intestinal epithelial tight junction barrier and cellular localization in Caco-2 cell monolayers (Liu et al., 2002; Wu ., 1996). This mechanism may similarly relate to glutamine-mediated modulation of intestinal barrier function in stressed animals and humans (Li ., 2004; DeMarco et al., 2003). that the intestine xylose absorptive ability improved from day 7 to 14 after weanling in both Gln supplement groups when compared to the control group. The results revealed that 1% Gln supplementation significantly (p<0.05) improved body weight. Weight gain, feed conversion ratio (FCR) when compared with the control, while 0.5 % Gln supplementation non- significantly (P>0.05). Improved broiler chock performance and the higher inclusion levels negative effect on broiler growth performance. Moreover, different levels Gln supplementation at different levels had heavier intestinal relative weights and longer intestinal villi (p<0.05) as compared with the control.
The results indicate that the addition of 1% Gln to the broiler chicks diet improved growth performance and may stimulate development of the gastrointestinal tract and immune response, while higher negative effects(soltan, 2009). The results indicated that dietary supplementation with 0.5 percent Gln may alleviate heat stress caused detrition in growth performance of broilers (Dia ,2009). It is known that glutathione production occurs in the liver; however, supplemental glutamine has been shown to increase gut glutathione production threefold (Cao ., 1998). This is the first report regarding the dietary Glutamine effect on performance and blood antioxidant status in broiler chickens under heat stress condition.
Although we didn’t assess the body or blood antioxidant indices at day 21 of age, the improved BWG of MG fed birds during the whole period possibly is related to their higher body antioxidant capacity due to Glutamine supplementation consumption. Although feeding 0.5 GLN improved the performance during the weekly and whole period but supplementation of higher level (1 percent) worsen the performance.
The supplementation glutamine effect significant on feed intake weekly, body weight gain whole during the broilers under heat stress but not effect significant feed intake and feed conversation ratio total during experimental and some blood antioxidant indices of broiler chickens under continuous heat stress condition.
The authors appreciate their thank to Mr. AmirMansorVatankhah (Department of Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran) for his help in determination of blood antioxidants indices.
This article was originally published in International Journal of Farming & Allied Sciences. Vol., 3 (12): 1213-1219, 2014.