Heat stress

The word ‘stress’ is commonly used to describe the detrimental effects of a variety of situations on the health and performance of poultry (Puron et al., Reference PURON, SANTAMARIA and SEGURA1994). It can be defined as the set of responses to external demands which force flocks to adapt to a new or abnormal situation (Puron et al., Reference PURON, SANTAMARIA and SEGURA1994; Virden and Kidd, Reference VIRDEN and KIDD2009). As a general term, stress is used to describe the sum of non-specific responses or defence mechanisms of the body when it is confronted with abnormal or extreme demands (Sahin et al., Reference SAHIN, SAHIN, KUCUK, HAYIRLI and PRASAD2009). Animals have been endowed with known zones of thermal comfort that vary by species, physiological status and the environmental effects of air flow, temperature and solar radiation (NRC, Reference NRC1981). When the environmental temperature exceeds the thermoneutral (TN) zone, the animal's body temperature increases as thermoregulatory reactions are exhausted. An animal is considered to be heat stressed when their temperature exceeds the upper critical level of the TN zone (Khan et al., Reference KHAN, NAZ, NIKOUSEFAT, TUFARELLI, JAVADANI, N. and LAUDADIO2011). Birds lose heat through the mechanisms of convection, conduction, radiation and evaporation as they do not possess sudorific glands (Puron et al., Reference PURON, SANTAMARIA and SEGURA1994) and are unable to sweat. To ameliorate this, birds try to lose heat by evaporative heat loss mechanism such as panting. When birds fail to use this mechanism to cool their bodies, then death is likely to occur (Ensminger et al., Reference ENSMINGER, OLDFIELD and HEINEMANN1990; Puron et al., Reference PURON, SANTAMARIA and SEGURA1994; Sahin et al., Reference SAHIN, SAHIN, KUCUK, HAYIRLI and PRASAD2009).

Birds usually have limited physical resources for growth, reproduction, response to environmental change and launching defence mechanisms (Khan et al., Reference KHAN, NAZ, NIKOUSEFAT, TUFARELLI, JAVADANI, N. and LAUDADIO2011). The adaptation to new challenges requires redistribution of body reserves of energy and protein at the cost of decreased growth and reproductive efficiency (Puron et al., Reference PURON, SANTAMARIA and SEGURA1994). Continuous stress causes fatigue and weakness and, consequently, affected birds are more likely to succumb to starvation and infectious diseases. During the initial phase of heat stress, postganglionic neurons and the medullary tissue of the adrenal glands release catecholamine including adrenaline and noradrenalin (Khan et al., Reference KHAN, NAZ, NIKOUSEFAT, TUFARELLI, JAVADANI, N. and LAUDADIO2011). The catecholamine prepares the birds for ‘fight or flight’ responses, triggering the rapid release of glucose. Following this, under the control of the hypothalamus, the glucocorticoid hormone, known as corticosterone, is released from the adrenal cortex (Puron et al., Reference PURON, SANTAMARIA and SEGURA1994). This hormone is responsible for the formation of glucose from body reserves of energy and protein. Finally, if the birds do not recover from the stress and the adaptability of the birds is not adequate, a third or exhaustive phase leads to fatigue of the homeostatic mechanisms and death of the birds (Puron et al., Reference PURON, SANTAMARIA and SEGURA1994).

Generally, stressors induce many physiological responses in poultry (Attia et al., Reference ATTIA, BOHMER and ROTH-MAIER2006; Attia et al., 2009). Panting results in a loss of CO₂ and an increase in blood pH. As the heat load increases, the rise in body temperature will result in tissue damage and the release of intracellular components into the blood. Creatine kinase (CK) is an intracellular enzyme and its plasma concentration has been shown to rise markedly during acute heat stress (Ostrowski-Meissner, Reference OSTROWSKI-MEISSNER1981; Whitehead and Keller, Reference WHITEHEAD and KELLER2003). Much of the tissue damage arises from lipid peroxidation which is elevated in acute heat stress. At the cellular level, heat stress causes an increase in the synthesis of heat shock proteins (HSP), also known as stress proteins. Increased HSP defends cells against damage and protects them against apoptosis (Wang and Edens, Reference WANG and EDENS1998; Coronato et al., Reference CORONATO, GIROLAMO, SALAS, SPINELLI and LAGUENS1999; Sahin et al., Reference SAHIN, SAHIN, KUCUK, HAYIRLI and PRASAD2009). During heat stress, HSP70, a type of heat shock protein located in the nucleus, binds to the RNA synthesis machinery, and prevents the synthesis and aggregation of abnormal proteins (Lee, Reference LEE1992). It is pertinent to note that the negative effects of heat stress depend upon exposure period, temperature, genotype and age of birds (Attia et al., Reference ATTIA, BOHMER and ROTH-MAIER2006; Lin et al., Reference LIN, JIAO, BUYSE and DECUYPERE2006; Attia et al., 2009).

For poultry, the TN zone ranges from 18 to 22°C. Within this zone, animals are expected to be comfortable, showing no problems associated with physical temperature regulation (NRC, Reference NRC1981; Ensminger et al., Reference ENSMINGER, OLDFIELD and HEINEMANN1990; Sahin et al., Reference SAHIN, SAHIN, KUCUK, HAYIRLI and PRASAD2009). Birds, during heat stress, have a greater challenge in maintaining homoeothermic body temperature, due to lack of sweat glands and higher body temperature (41.5°C) compared to other domestic animals (Ensminger et al., Reference ENSMINGER, OLDFIELD and HEINEMANN1990; Sahin et al., Reference SAHIN, SAHIN, KUCUK, HAYIRLI and PRASAD2009). Heat stress is one of the major stressors in poultry production and produces a wide range of physiological alterations, the nature of which depends upon the degree of heat stress imposed (Whitehead and Keller, Reference WHITEHEAD and KELLER2003). Typical responses include increases in plasma concentrations of corticosteroids, protein, glucose and heterophil to lymphocyte ratio (H/L), reduction in potassium as well as the relative weights of the adrenal glands, bursa, spleen and thyroid glands (Pardue et al., Reference PARDUE, THAXTON and BRAKE1985; Kutlu and Forbes, 1993; Whitehead and Keller, Reference WHITEHEAD and KELLER2003). To minimise the heat load, laying birds will reduce feed intake and production will decrease. High ambient temperatures have a detrimental effect on growth in broilers (Smith, Reference SMITH1974; Yalcin et al., Reference YALCIN, OZKAN, TURKMUT and SIEGEL2001; Sahin et al., Reference SAHIN, SAHIN, KUCUK, HAYIRLI and PRASAD2009). Heat stress also causes consistent reductions in egg weight and shell thickness in laying hens, while it has been associated with undesirable meat characteristics in rapidly growing birds (Emery et al., Reference EMERY, VOHRA, ERNST and MORRISON1984; Sandercock et al., Reference SANDERCOCK, HUNTER, NUTE, MITCHELL and HOCKING2001; Wolfenson et al., Reference WOLFENSON, BACHRACH, MAMAN, GRABER and ROZENBOIM2001). Several studies have shown that high ambient temperatures may adversely influence the immune system of birds (Henken et al., Reference HENKEN, SCHAARSBERG and NIEUWL1982; Donker et al., Reference DONKER, NIEUWLAND and VAN DER ZIJPP1990; Sahin et al., Reference SAHIN, SAHIN, KUCUK, HAYIRLI and PRASAD2009). Heat stress also decreases minerals (e.g. Fe, Zn and Cr) and vitamins (e.g. vitamin C, A, and E) in the blood and increases their excretion from the body, leading to deficiencies of these components in blood and tissues (McDowell, Reference MCDOWELL1989; Siegel, Reference SIEGEL1995; Klasing, Reference KLASING1998; Sahin et al., Reference SAHIN, SAHIN and YARALIOGLU2002; Sahin et al., Reference SAHIN, SAHIN, KUCUK, HAYIRLI and PRASAD2009). The mortality of birds proportionally increases with increases in temperature and it is especially high when marketing age is drawing near (Arjona et al., Reference ARJONA, DENBOW and WEAVER1990).

Ascorbic acid

Vitamin C or L-ascorbic acid is an antioxidant which is normally synthesized by the chicken (Khan, Reference KHAN2011). It is a simple compound which was initially detected in the mammalian adrenal gland. It was given the name hexuronic acid and later it was also known as cevitaminic acid scorbutamin and ant scorbutamin vitamin (Elkheir et al., Reference ELKHEIR, MOHAMMED, AHMED and ABDELGADIR2008). Vitamin C has an important metabolic role as a result of its reducing properties and functions as an electron donor. The biosynthesis of ascorbic acid in birds and mammals occurs in the liver and kidneys, however in chickens, biosynthesis mainly occurs in the kidneys (Elkheir et al., Reference ELKHEIR, MOHAMMED, AHMED and ABDELGADIR2008). Sugars such as glucose, fructose and mannose serve as precursors for the synthesis of ascorbic acid. Ascorbic acid can donate two electrons and in the process is converted to dehydro-L-ascrobic acid. It takes part in redox and hydroxylation pathways, acting as a cofactor for cytochrome P450-dependant hydroxylases in some of the reactions. Ascorbic acid is not an essential nutrient for avian species because they possess enzyme gulonolactone oxidase which is required for the biosynthesis of this vitamin and is lacking in humans and some other species (Lin et al., Reference LIN, JIAO, BUYSE and DECUYPERE2006; Khan, Reference KHAN2011). Hence no recommended requirement for ascorbic acid has been established by NRC for birds (NRC, Reference NRC1994). However it has been recommended in poultry feeds as a supplement to alleviate stress, on the assumption that during stress the requirements may exceed the synthesizing ability (Gous and Morris, Reference GOUS and MORRIS2005). Physiological stressors like heat, disease or overcrowding may augment the chicken's requirement for ascorbic acid (Nockels, Reference NOCKELS1984). Moreover, high ambient temperatures impair the absorption of ascorbic acid and increase the dietary requirement of this vitamin (Klasing, Reference KLASING1998).

Chickens require ascorbic acid for amino acid and mineral metabolism, as well as for the synthesis of hormones like testosterone (McDowell, Reference MCDOWELL1989). Ascorbic acid is involved in the production of white blood cells and thus enhances the immune status of birds. Recently, there has been considerable interest in a possible nutritional role of ascorbic acid on the basis that (a) endogenous synthesis may not be adequate to address the full needs of poultry at all times and (b) the requirement of this vitamin may increase during heat stress (Lin et al., Reference LIN, JIAO, BUYSE and DECUYPERE2006). High environmental temperatures may increase the requirement of ascorbic acid, decrease the biosynthesis of this vitamin and affect the endocrine system responsible for the retention and proper metabolic functioning of this vitamin. It has been reported that birds under heat stress can recognize feed supplemented with ascorbic acid and adjust their need according to their requirements. Kutlu and Forbes (1993) showed that broilers under heat stress utilized more supplemented feed when ascorbic acid fortified feed was coloured. When the condition was reversed to TN environment, the selection was changed.

Growth, performance and productivity

It has been shown that heat stress decreases feed intake, body weight and egg production, egg weight and quality (Ciftci et al., Reference CIFTCI, ERTAS and GULER2005). Reductions in feed intake are proportional to the severity and length of exposure to heat stress (Mashaly et al., Reference MASHALY, HENDRICKS, KALAMA, GEHAD, ABBAS and PATTERSON2004). Hurwitz et al. (Reference HURWITZ, WEISELBERG, EISNER, BARTOV, RIESENFELD, SHARVIT, NIV and BORNSTEIN1980) showed that appetite is decreased as a primary response to high temperatures. Decreased body weight is attributed to the reductions in feed intake as a result of high ambient temperatures. Mckee et al. (Reference MCKEE, HARRISON and RISKOWSKI1997) suggested that due to heat stress, feed intakes are lowered in birds; hence birds do not consume adequate amounts of ascorbic acid. Consequently, supplementing drinking water with ascorbic acid may be a more suitable choice because unlike feed intake, water consumption increases during high ambient temperatures. A high fat diet may increase the performance of poultry in the presence of ascorbic acid. A high fat diet would improve an animal's heat tolerance because fat metabolism produces a lower heat increment. Since net energy for production is equal to metabolizable energy minus the heat increment, more energy would be available for maintenance and production performance (Mckee et al., Reference MCKEE, HARRISON and RISKOWSKI1997).

At high temperatures, thyroid activity is arrested, which in turn affects metabolic rate, oxygen consumption, feed intake and growth rate. Supplemental ascorbic acid has been shown to attenuate these negative responses in poultry under heat stress by increasing oxygen consumption, feed intake and growth rate (Mckee et al., Reference MCKEE, HARRISON and RISKOWSKI1997). Metabolizable energy is devoted to the maintenance of relatively constant internal biological conditions. During heat stress birds divert metabolizable energy from production to performance to the maintenance of homeothermy (Mckee et al., Reference MCKEE, HARRISON and RISKOWSKI1997). Heat-exposed birds decrease their feed consumption to reduce their thermogenic effect associated with nutrient absorption, assimilation and utilization. In addition, reduced blood flow to the gastrointestinal tract (GIT) during heat-induced peripheral vasodilatation may impair nutrient absorption (Mckee et al., Reference MCKEE, HARRISON and RISKOWSKI1997). Decreased feed intake and reduced blood flow to the GIT may exacerbate metabolic energy input. These factors lead to depressed performance characteristics in poultry exposed to heat stress (Mckee et al., Reference MCKEE, HARRISON and RISKOWSKI1997).

Sahin et al. (Reference SAHIN, ONDERCI, SAHIN, GURSU and KUCUK2003) concluded that supplementation of ascorbic acid may offer enhanced protection to heat stress-related depression in Japanese quails. The physiological and performance impact of ascorbic acid has been ascribed to an increase in the bird's appetite resulting in increased food intake. In broilers, Sahin and Kucuk (Reference SAHIN and KUCUK2001) reported that supplemental ascorbic acid (250 mg/kg of diet) increased performance and yielded better carcass quality in birds reared under heat stress conditions (32°C). Sahin et al. (Reference SAHIN, ONDERCI, SAHIN, GURSU and KUCUK2003) reported significant negative effects on live weight gain, feed intake, efficiency and carcass traits in quails exposed to high ambient temperatures (34°C). Attia et al. (2009) reported that at a level of 250 mg/kg, ascorbic acid relieved the birds from the negative effects of heat stress and increased feed intake, protein digestibility, dressing percentage and feed conversion ratio (FCR). The improved performance of heat stressed broiler chicks in response to ascorbic acid has been shown to correspond with lower plasma corticosterone concentrations (Pardue et al., Reference PARDUE, THAXTON and BRAKE1985; Kutlu and Forbes, 1993). Sahin and Kucuk (Reference SAHIN and KUCUK2001) recorded better body weight and feed intake in heat stressed broiler chickens in response to 200 mg/kg of ascorbic acid. Kutlu and Forbes (1993) reported that broiler chickens reared under heat stress (36°C) experienced a reduced growth rate, feed intake and FCR, however, with supplementation of ascorbic acid at the level of 250 mg/kg of diet, these parameters improved. Coskun and Kutlu (Reference COSKUN and KUTLU1997) reported that ascorbic acid supplementation (250 mg) improved carcass yield in heat stressed broiler chickens. Ciftci et al. (Reference CIFTCI, ERTAS and GULER2005) found that supplementation of ascorbic acid (200 mg/kg) in combination with vitamin E (125 mg/kg) significantly improved feed efficiency and body weight gain in laying hens exposed to heat stress. Njoku (Reference NJOKU1986) reported a positive response in body growth and feed intake of heat stressed broilers to supplemental ascorbic acid (200 mg/kg of diet). Elkheir et al. (Reference ELKHEIR, MOHAMMED, AHMED and ABDELGADIR2008) found superior performance in weight gain at the level of 250 mg/kg of ascorbic acid at temperatures as high as 42°C in broilers. McDowell (Reference MCDOWELL1989) , Kutlu and Forbes (1993) and Sahin et al. (Reference SAHIN, SAHIN and YARALIOGLU2002) reported that ascorbic acid reduces the synthesis of corticosteroid hormones and adenocorticotropic hormone (ACTH) in birds, and improves performance thereafter.

It is well known that egg production decreases when ambient temperature goes above the TN zone. Mashaly et al. (Reference MASHALY, HENDRICKS, KALAMA, GEHAD, ABBAS and PATTERSON2004) concluded that egg production is inversely related to high temperature. Whitehead et al. (Reference WHITEHEAD, BOLLENGIER-LEE, MITCHELL and WILLIAMS1998) and Kirunda et al. (Reference KIRUNDA, SCHEIDELER and MCKEE2001) found that egg production in White Leghorns decreased when they were exposed to high environmental temperatures. The decrease in egg production has been ascribed to the decreased feed consumption, reducing the availability of essential nutrients for egg production (Mashaly et al., Reference MASHALY, HENDRICKS, KALAMA, GEHAD, ABBAS and PATTERSON2004). It has been reported that exposure to high heat decreased plasma protein concentration and plasma calcium (Ca), both of which are required for egg formation (Mashaly et al., Reference MASHALY, HENDRICKS, KALAMA, GEHAD, ABBAS and PATTERSON2004). During heat stress, corticosterone increases conversion of norepinephrine to epinephrine, which induces degradation of ovarian follicles (Ciftci et al., Reference CIFTCI, ERTAS and GULER2005). Exposure to high heat also decreases the egg weight, shell weight, shell thickness and specific gravity (Mashaly et al., Reference MASHALY, HENDRICKS, KALAMA, GEHAD, ABBAS and PATTERSON2004). High ambient temperatures impair the synthesis and release of vitellogenine necessary for yolk formation (Ciftci et al., Reference CIFTCI, ERTAS and GULER2005). The deteriorated egg shell quality under heat stress has been attributed to a reduction in Ca uptake by the duodenal epithelium (Mashaly et al., Reference MASHALY, HENDRICKS, KALAMA, GEHAD, ABBAS and PATTERSON2004). Smith (Reference SMITH1974) concluded that the decline in egg weight at high temperatures is due mainly to the effect of heat stress per se and not to the effect of diminished feed intakes. Sahin and Sahin (Reference SAHIN2001) reported that at low ambient temperatures (6.2°C), egg weight, specific gravity, egg shell thickness, egg shell weight and Haugh unit were improved in response to 250 mg/kg of ascorbic acid in the diet. In laying hens, it was postulated that ascorbic acid stimulates 1,25-dihydroxycholecalciferol and increases calcium mobilization from bones, suggesting that ascorbic acid has an important role in egg shell formation (Sahin and Sahin, Reference SAHIN2001). Nowaczewski and Kontecka (Reference NOWACZEWSKI and KONTECKA2005) recorded higher egg production and egg fertility in aviary pheasants, supplemented with 200 mg/kg ascorbic acid, reared at a temperature range of 25-30°C. Ciftci et al. (Reference CIFTCI, ERTAS and GULER2005) found that supplementation of ascorbic acid (200 mg/kg) in conjunction with vitamin E (125 mg/kg) significantly improved egg production, egg weight, specific gravity, shell thickness and percentage of yolk in laying hens exposed to heat stress (32°C).

Nutrient digestibility

The effect of heat stress in birds involves two key factors: feed intake and metabolic modifications (Gous and Morris, Reference GOUS and MORRIS2005; Attia et al., 2009). During heat stress, animal cells attempt to hold water by accumulating K ions. The increase K and other ions generally induce negative effects on the metabolic process (Attia et al., 2009). High ambient temperatures are known to decrease nutrient digestibility (Wallis and Balnave, Reference WALLIS and BALNAVE1984; Sahin and Kucuk, Reference SAHIN and KUCUK2001). Wallis and Balnave (Reference WALLIS and BALNAVE1984) found that the digestibility of amino acids was decreased by high environmental temperatures in broilers. Similar conclusions were drawn by Larbier et al. (Reference LARBIER, CHAGNEAU and GERAERT1993). Likewise, Zuprizal et al. (Reference ZUPRIZAL, CHANNEAU and GERAERT1993) demonstrated that true digestibility of protein and amino acids from two different sources decreased as the temperature increased from 21°C to 32°C. Bonnet et al. (Reference BONNET, GERAERT, LESSIRE, CARRE and GUILLAUMIN1997) reported that exposure to heat stress, decreased the digestibility of protein, fat and starch. The increase in dressing percentage in birds is parallel to protein digestibility (Attia et al., 2009). Hai et al. (Reference HAI, RONG and ZHANG2000) concluded that low digestibility of feed is due to the decline in the activities of trypsin, chymotrypsin and amylase at high temperatures.

Heat stress not only reduces feed intake but has also been reported to reduce the digestibility of different components of the diet (Bonnet et al., Reference BONNET, GERAERT, LESSIRE, CARRE and GUILLAUMIN1997; Mashaly et al., Reference MASHALY, HENDRICKS, KALAMA, GEHAD, ABBAS and PATTERSON2004; Attia et al., 2009). Ascorbic acid induces changes in metabolism, which is the basis of positive responses of supplementing this vitamin in poultry diet during heat stress. Because ascorbic acid has protective roles on pancreatic tissues to oxidative stress, it may help the pancreas to function properly, thus improving the digestibility of nutrients (Attia et al., 2009). Sahin and Kucuk (Reference SAHIN and KUCUK2001) observed that the digestibility of dry matter (DM), crude protein (CP), organic matter (OM) and ether extract (EE) were greater with high dietary ascorbic acid (200 mg/kg of diet) in Japanese quails, reared under chronic heat (34°C). Low ambient temperatures have been reported to suppress nutrient digestibility, which is restored by dietary supplementation of ascorbic acid (Sahin and Sahin, Reference SAHIN2001). Sahin et al. (Reference SAHIN, ONDERCI, SAHIN, GURSU and KUCUK2003) demonstrated that supplementation of ascorbic acid increased the retention of nitrogen, ash, and Ca as compared to heat stressed controls. In another study, Sahin et al. (Reference SAHIN, SAHIN and YARALIOGLU2002) studied the effect of low ambient temperatures (6.8°C) on the nutrient digestibility of laying hens and concluded that the digestibility of DM, OM, CP and EF increased by supplementation of ascorbic acid (250 mg/kg of diet). Sahin and Sahin (Reference SAHIN2001) observed that at low ambient temperatures (6.2°C), the digestibility of DM, ash, OM, CP and EE were higher with higher levels of ascorbic acid (125 versus 250 mg/kg) in laying hens. Similarly Sahin (Reference SAHIN2001) reported a decrease in the utilization of DM, CP and EE in laying hens kept at low temperatures (6.9°C). Ciftci et al. (Reference CIFTCI, ERTAS and GULER2005) suggested that oxidative lesions result in conformational modification of protein which induces inhibition of pancreatic enzymes or dietary protein resistance to digestion. Consequently, the presence of ascorbic acid could partially interfere with oxidative protein denaturation and would improve the digestibility of nutrients and feed efficiency.

Immunity

Numerous studies have demonstrated the effects of heat stress on immunity in chickens (Amedy et al., Reference AMEDY, TAYEB and YOKHANA2011). Kadymov and Aleskerov (Reference KADYMOV and ALESKEROV1988) concluded that high temperatures cause inhibition of the synthesis of T and B lymphocytes and suppression of phagocytic activity of blood leukocytes. Mashaly et al. (Reference MASHALY, HENDRICKS, KALAMA, GEHAD, ABBAS and PATTERSON2004) reported a decline in white blood cells (WBC) after exposure to heat which indicates that heat stress can reduce both the number and activities of leukocytes. Mashaly et al. (Reference MASHALY, HENDRICKS, KALAMA, GEHAD, ABBAS and PATTERSON2004) reported decreases in heterophil to lymphocyte (H/L) ratios in White Leghorn hens. The H/L ratio has been used as a reliable indicator of stress in birds (Gross and Siegel, Reference GROSS and SIEGEL1983; Mashaly et al., Reference MASHALY, HENDRICKS, KALAMA, GEHAD, ABBAS and PATTERSON2004). McFarlane and Curtis (Reference MCFARLANE and CURTIS1989) supported the view that the H/L ratio increased under heat stress in broiler chicks. Mashaly et al. (Reference MASHALY, HENDRICKS, KALAMA, GEHAD, ABBAS and PATTERSON2004) reported that humoral immunity of the birds from heat stress is depressed. Zulkifli et al. (Reference ZULKIFLI, NORMA, ISRAF and OMAR2000) showed that heat stress caused a reduction in antibody synthesis. This reduction has been ascribed to increases in inflammatory cytokines under stress, which stimulates the hypothalamic production of corticotropin releasing factor (Sapolsky et al., Reference SAPOLSKY, RIVIER, YAMAMOTO, PLOTSKY and VALE1987; Mashaly et al., Reference MASHALY, HENDRICKS, KALAMA, GEHAD, ABBAS and PATTERSON2004). Furthermore, heat stress has been known to depress T-helper cytokines, which are important for antibody production (Lebman and Coffman, Reference LEBMAN and COFFMAN1988; Mashaly et al., Reference MASHALY, HENDRICKS, KALAMA, GEHAD, ABBAS and PATTERSON2004).

Ascorbic acid is an important antioxidant in biological system and could control the adverse effect of heat stress on immunological competence (Gous and Morris, Reference GOUS and MORRIS2005; Lin et al., Reference LIN, JIAO, BUYSE and DECUYPERE2006). Ascorbic acid plays a major role in enhancing the immune system. Studies have demonstrated that ascorbic acid is required for the systemic availability of immunoglobulins and interferons (Bendich, Reference BENDICH1990). Furthermore, CD8+ and IgM cells are increased as a result of ascorbic acid effects (Wu et al., Reference WU, DORAIRAJAN and LIN2000). Heat stress has also been shown to suppress the weights of the bursa and spleen (Pardue et al., Reference PARDUE, THAXTON and BRAKE1985). Under heat stress, ascorbic acid has been shown to enhance immunological profiles. Attia et al. (2009) reported that at a level of 250 mg/kg, ascorbic acid relieved birds from the negative effects of heat stress and improved humoral immune competence to sheep red blood cells (SRBC). Bashir et al. (Reference BASHIR, MUNIR, SAEED, RAZ and RAZA1998) recorded elevated haemagglutination titre in heat stressed broilers vaccinated against Newcastle disease (ND) in response to supplemental ascorbic acid. Similarly, Pardue et al. (Reference PARDUE, THAXTON and BRAKE1985) found elevated haemagglutination response to SRBC in heat stressed chickens in the presence of supplemented ascorbic acid. Some studies have also shown the efficacy of ascorbic acid in conjunction with vitamin E on immunological function in birds under heat stress. Puthpongsiriporn et al. (Reference PUTHPONGSIRIPORN, SCHEIDELER, SELL and BECK2001) observed that the combination of vitamin E (65 mg/kg diet) and ascorbic acid (1000 g/l drinking water) enhanced lymphocyte proliferative responses to concanavalin and lipopolysacheride stimulation.

Antioxidants

Reactive oxygen species (ROS) are generated at the cellular level during normal body function. However, high ambient temperatures have been shown to augment the production of free radicals in body fluids and tissues. Although low levels of ROS are necessary for many biochemical processes. However, the increased production of ROS may damage biological macromolecules and disruption of normal cell metabolism. Physiologically, the body has its own system of antioxidants to neutralize the effect of ROS. High ambient temperatures deplete such reserves of antioxidants and causes oxidative stress. Ascorbic acid can act as an antioxidant by two ways. Firstly, it can be readily oxidised to dehydroascrobic acid, which is reversible, and form the basis of many physiological activities. This vitamin also forms ascorbate radical and destroys hydroxyl, singlet oxygen and superoxide radicals. In this capacity, it may act synergistically with other antioxidant enzymes including superoxide dismutase, glutathione peroxidase and catalase (Lin et al., Reference LIN, JIAO, BUYSE and DECUYPERE2006). Ascorbic acid is also involved in the antioxidant system of vitamin E, by sparing it for neutralizing other radicals, or converting the oxidized form of α-tocopherol back to α-tocopherol. Free radicals are formed during the conditions of heat stress, reducing membrane integrity because of lipid peroxidation in the cell membrane (Klasing, Reference KLASING1998; Sahin et al., Reference SAHIN, ONDERCI, SAHIN, GURSU and KUCUK2003). It was previously reported that the concentration of ascorbic acid in the serum and liver decreases during heat stress (Sahin et al., Reference SAHIN, SAHIN and YARALIOGLU2002). Sahin et al. (Reference SAHIN, ONDERCI, SAHIN, GURSU and KUCUK2003) reported significant negative effects on antioxidant status in quails exposed to high ambient temperatures (34°C). It has been shown that concentrations of malondialdehyde (MDA), an indicator of lipid peroxidation in the serum, liver, kidney and heart decreases with supplementation of ascorbic acid in quails reared under heat stress (Sahin et al., Reference SAHIN, ONDERCI, SAHIN, GURSU and KUCUK2003). In the same study, it was also demonstrated that ascorbic acid increased the serum concentration of other antioxidant vitamins. Sahin et al. (Reference SAHIN, SAHIN and YARALIOGLU2002) studied the effect of 250 mg of ascorbic acid per kg of diet and the same amount of VE both alone or in combination on serum MDA, in laying hens reared at 25 to 35°C and concluded that separately or in combination, ascorbic acid and VE increased serum ascorbic acid and VE and decreased MDA concentration. In another study, Sahin et al. (Reference SAHIN, SAHIN and YARALIOGLU2002) studied the effect of low ambient temperature (6.8°C) on serum MDA in response to 250 mg of ascorbic acid per kg of diet in laying hens and concluded supplementation not only decreased serum MDA but also increased the serum concentration of ascorbic acid and VE. Puthpongsiriporn et al. (Reference PUTHPONGSIRIPORN, SCHEIDELER, SELL and BECK2001) found that thiobarbarbituric acid (TBA), product of lipid peroxidation, in eggs yolk and plasma were improved by supplementation of VE in heat stress hens, however, ascorbic acid had no effect when supplemented at the level of 1000 mg/l of water in combination with VE. Ascorbic acid is found in negligible amounts compared to other yolk vitamins and it is possible that supplementation of ascorbic acid did not express an additive effect to VE supplementation in yolk because of very low levels deposited in the yolk. Hoehler and Marquardt (Reference HOEHLER and MARQUARDT1996) suggested that the antioxidant effect of ascorbic acid may be less strong than the supplementation of VE in vivo.

Male reproduction

The effect of heat stress on semen quality is debatable among many researchers due to conflicting results (McDaniel et al., Reference MCDANIEL, HOOD and PARKER2004). Clark and Sarakoon (Reference CLARK and SARAKOON1967) found no effect of heat stress on semen characteristics of White Leghorn males, under cyclic ambient temperature, ranging from 21 to 38°C. However, Joshi et al. (Reference JOSHI, PANDA and JOSHI1980) found decreased semen volumes, sperm concentrations, live sperm and sperm motility at 32°C. McDaniel et al. (Reference MCDANIEL, HOOD and PARKER2004) reported that the percentage of dead sperm increased during the heat stress period and then decreased immediately upon removal of heat stress. McDaniel et al. (Reference MCDANIEL, BRAMWELL, WILSON and HOWARTH1995) found that heat stress did not affect semen quality in male broiler breeders. Sever heat stress also depresses fertility along with sperm motility and viability (McDaniel et al., Reference MCDANIEL, HOOD and PARKER2004). Sperm motility is very important for successful fertilization, since sperm from heat stressed males may be unable to reach the sperm storage tubules in the uterovaginal junction due to impaired motility (Khan et al., Reference KHAN, RAHMAN, JAVED and MUHAMMAD2012a; Reference KHAN, LAUDADIO and TUFARELLI2012b). McDaniel et al. (Reference MCDANIEL, HOOD and PARKER2004) suggested that during heat stress, the DNA of spermatozoa is damaged which results in abnormal mitotic division during spermatogenesis. It would yield abnormal spermatozoa which may be unable to penetrate the ovum to complete the fertilization process.

Ascorbic acid has been reported to increase fertility and semen quality under heat stress. Ascorbic acid at a rate of 200 mg/kg of feed is also beneficial for reproductive aviary pheasants housed under heat stress (Nowaczewski and Kontecka, Reference NOWACZEWSKI and KONTECKA2005). Dobrescu (Reference DOBRESCU1987) documented that 150 mg/kg of ascorbic acid fed to turkey toms increased semen quality. Monsi and Onitchi (1991) reported that feeding ascorbic acid at the levels of 125 and 250 ppm increased semen volume, sperm concentration, sperm per ejaculate and number of motile sperm. Peebles and Brake (Reference PEEBLES and BRAKE1985) found that the fertility of male and female broiler breeders improved in response to 150 mg/kg of dietary ascorbic acid. However, McDaniel et al. (Reference MCDANIEL, HOOD and PARKER2004) found that excessive administration (500 and 1000 mg/kg) of ascorbic acid resulted in depression of fertility over each day the males were heat stressed and concluded that only specific levels of ascorbic acid are beneficial to heat stressed poultry. According to McDaniel et al. (Reference MCDANIEL, HOOD and PARKER2004) the results seen with supplementation of ascorbic acid recorded by different researchers are very inconsistent. This may be due to oxidative degradation of this compound, leading to false results (Mckee and Harrison, Reference MCKEE and HARRISON1995).

In conclusion, heat stress causes significant reductions in performance, productivity, nutrient digestibility, immune status, antioxidant profile and male fertility. These implications result in increased disease incidence and economic losses in poultry operations. Because of its significant role in poultry production and performance, ascorbic acid is a very important component of poultry feed during times of heat stress.