Effects of different heat stress periods on various blood and meat quality parameters in young Arbor Acer broiler chickens

Heat stress can influence muscle metabolism and meat quality (Wood and Richards 1975 ; Sandercock et al. 2001 ; Aksit et al. 2006 ; Lu et al. 2007 ; Gregory 2010 ). Especially in broiler chickens, the acute heat stress that provokes a sufficient adrenaline response to affect meat quality is very severe and is near the lethal limit (Nagle et al. 2000 ; Lowe et al. 2002 ). Stressful conditions that lead to the depletion of muscle glycogen reserves before slaughter may lead to higher ultimate pH (pHu) values in meat and result in low residual levels of glucose (Bray et al. 1989 ). Increases in the pHu of m. longissimus thoracis from a normal value of 5.5 to an intermediate value of approximately 6 are associated with increased Warner–Bratzler shear force values (Watanabe et al. 1996 ; Purchas et al. 1999 ). The post-mortem metabolism of intramuscular energy stores (e.g., glycogen) plays a primary role in changing pH value of muscle, which promotes different quality attributes. Loss of blood circulation at death causes post-mortem anaerobic glycolysis, resulting in an accumulation of lactic acid. This accumulation causes the pH of the muscle to decline after death. Large amounts of muscle glycogen at the time of death result in a low pHu, consequently resulting in a pale color and lower water-holding capacity (WHC). Pale, soft, and exudative (PSE) broiler breast meat is comparable to PSE pork (Barbut 1998 ). Compared with other species, broiler chickens are more sensitive to high ambient temperatures because of their normally elevated body temperature, rapid metabolism and lack of sweat glands (Geraert et al. 1993 ). Furthermore, modern broiler breeds are more susceptible to heat stress than earlier genotypes. It is claimed that heat-induced suppression of growth is now being seen at lower ambient temperatures in commercial strains (Deeb et al. 2002 ). In addition, raised ambient temperature (>30°C) from 4 wk of age up to the point of marketing has been shown to reduce growth performance as a result of decreased feed intake, growth rate and feed utilization of broilers (Cahaner and Leenstra 1992 ; Cooper and Washbrun 1998 ). Moreover, exposure of chickens to high temperature (>32°C) tends to reduce nutrient digestion as a result of decreasing blood flow to the digestive system. In addition, fast-growing strains have lower survival during heat stress (Yalcin et al. 2001 ).

Exposure to high ambient temperatures has been recognized as one of the main environmental factors that influence meat quality (Northcutt et al. 1994 ; Aksit et al. 2006 ; Lu et al. 2007 ). Commercial transportation and simulations of the conditions of acute heat stress during transportation have been shown to induce skeletal muscle damage (myopathy) in broiler chickens (Mitchell and Sandercock 1995 ) and pigs (Yu et al. 2009 ). Heat-stress-induced ante-mortem alterations in muscle membrane permeability and associated changes in muscle metabolism in broilers may influence post-mortem meat quality. Hyperthermia-associated myopathy is characterized by an increase in the plasma activity of the skeletal muscle-derived isoenzyme creatine kinase (CK), reflecting Ca-mediated alterations in muscle membrane integrity (Mitchell and Sandercock 1997 ; Sandercock et al. 2001 ). Similar increases in plasma CK activity have been reported for hyperthermia-associated stress conditions in pigs and have been shown to be associated with detrimental effects on meat quality (Shibata 1996 ).

Variations and defects in color in poultry have long been recognized as major problems, with most of the evidence for the mechanism pointing towards preslaughter acute or short-term heat stress leading to PSE-like changes in broilers (Northcutt et al. 1994 ; Sandercock et al. 2001 ; Ziober et al. 2010 ). However, little information is available on the characteristics of PSE-quality broiler breast meat, and few reports have addressed both ante- and post-mortem responses within the same study. From a commercial perspective, understanding the cause of this condition is clearly necessary. A series of changes in the neuroendocrine system of animals is well known to occur after heat stress. Furthermore, certain clinical symptoms, such as high body temperature, low blood pressure and inordinate breath rate, indicate that animals are stressed. However, these effects have been shown to be species-dependent.

In the present study, plasma CK, glutamic-pyruvic transaminase (GPT), and glutamic-oxaloacetic transaminase (GOT) were investigated as possible parameters for evaluation of stress in broilers. The aim of the present study was to characterize skeletal muscle responses to acute heat stress exposure for various periods of ante-mortem time in broiler chickens and assess the relationship between such responses and the possible changes in broiler meat quality.

Statistical analysis of the differences between groups was performed by one-way analysis of variance (ANOVA) using the Statistical Package for Social Sciences (SPSS version 11.5). The mean value of the control group was compared with each experimental group using the Least Significant Difference test. A regression test was also conducted to assess the Pearson correlation coefficients based on the different parameters. Differences were regarded as significant at P< 0.05.

Color was measured with a Minolta surface spectrocolorimeter (Model CR200; D65 illuminant, 8-mm viewing diameter). Breast muscles were exposed to air for at least 10 min at 15°C before color measurement. Lightness, redness, and yellowness values at three locations in the thickest part of the ventral surface in the middle of the pectoralis muscles were recorded and averaged.

Frozen chicken breast fillets stored at −20°C were thawed in pre-warmed in cooking bags before being weighed and cooked. The fillet was immersed in a 70°C water bath. After the internal temperature reached 70°C, the fillet was cooked for approximately 45 min. The temperature was measured with a cooking thermometer inserted into the thickest part of the breast fillet. After cooking, the samples were cooled in an ice bath for 30 min and then reweighed. Cooking loss was subsequently calculated. The shear force values, which account for meat tenderness, were determined for the cooked fillets (1×2×0.5 cm) using a Warner–Bratzler shearing device. The cooking loss was calculated based on the weight of the fillets before and after cooking according to the following formula:

A cored sample (diameter, 2 cm; thickness, 0.5 cm) of the pectoralis muscle was collected and weighed. Subsequently, the core was compressed by a force meter with 35 kg of force for 5 min. The samples were reweighed, and the expressible moisture was calculated based on the weights before and after compression according to the following formula:

The pH of meat was measured by the direct probe method with an Orion pH meter (HANNA-HI9025, Italy) equipped with a spear-shaped Mettler-Toledo combination pH electrode. The probe was inserted into the center of the muscle at a distance of 0.5 to 1 cm below the surface. The pH values of the pectoralis were measured at 15 min (pHi) and 24 h (pHu) post-mortem (Sandercock et al. 2001 ). The pH meter was standardized by a two-point calibration against standard buffers at pH 4.0 and 7.0.

The activities of plasma CK, GPT, and GOT were assessed using a commercial kit (Nanjing Jiancheng, China) modified for use with a multi-well plate spectrophotometer as previously described by (Sun et al. 2005 ). Plasma insulin was detected following the method described by McMurtry et al. ( 1983 ). Glucagon was determined as previous described by McMurtry et al. ( 1996 ). The radioimmunoassay kits of plasma insulin and glucagon were provided by (Ruiqi biotech CO., LTD Shanghai, China).

One-day-old male Arbor Acer (AA) broiler chickens (=120) were obtained from the Nanjing Changjiaying Commercial Fowl Company (China). The birds were housed in large coops (20 birds per coop) placed in a climate-controlled chamber. The birds were allowed to acclimate to their new housing conditions and recover from environmental stress for 4 wk. During this period, the broilers were reared according to previous research (Sandercock et al. 2001 ). The total broiler population was vaccinated against Newcastle disease and infectious bursal disease on days 7 and 14, respectively. The room temperature was maintained at 35°C during the first week. As the chickens grew, the temperature was gradually reduced by 3°C every 7 d until to 22°C was reached. This temperature was maintained by controlled ventilation and heating until day 30. Subsequently, the room temperature was increased rapidly from 22±1°C to 37±1°C. AA broilers (=120) were then randomly divided into six groups (20 per group) defined by the duration of heating: 0 (control), 1, 2, 3, 5, and 10 h of heat stress. Temperature was monitored in the center of each coop. The birds were given access to a commercial broiler feed and water ad libitum during the heat stress period. At the end of the heat stress period, blood samples (10 mL) were obtained from the carotid jugular vein by syringe, transferred to blood collection tubes that contained heparin anti-coagulant (50 IU mL), and immediately chilled on ice. The birds were then sacrificed by cervical dislocation. The plasma samples for subsequent hormone and enzyme determinations were obtained by centrifuging whole blood at 1500×for 5 min, frozen, and then stored at −20°C. Following exsanguination, the birds were manually eviscerated, and part of the pectoralis was collected 15 min after slaughter for the measurement of pHi. A separate part of the pectoralis was stored at 4°C for 24 h until sampling was conducted for the measurement of pHu, meat color, and WHC.

The Pearson correlation coefficients between various the quality parameters of the pectoralis are presented in Table 3 . The Pearson correlation coefficient for the relationship between pHi and pHu was 0.560 and 0.515 for the relationship between cooking loss and expressible moisture, exhibiting a significant positive correlation for these parameters. Moreover, the Pearson correlation coefficients for the relationship between pHi and cooking loss, expressible moisture,value,value, and shear force value were −0.341, −0.336, −0.329, 0.600, and −0.288, respectively.

The carcass and meat quality characteristics are presented in Table 2 . As a consequence of the heat stress treatment, the values of both pHi and pHu in the pectoralis were lower in heat-stressed chickens than in control chickens (0.01; Table 2 ). Significant decreases in pHi in the pectoralis after 1 h of heat stress and pHu after 2 h of heat stress were observed (0.01). Upon heat stress, both the pHi and pHu values of the pectoralis gradually decreased. The cooking loss and expressible moisture of the pectoralis in the heat-stressed groups gradually increased compared with controls ( Table 2 ). However, significant increases in cooking loss and expressible moisture were observed after 3 h of heat stress (0.01). The shear force values increased (0.05) in the groups exposed to acute heat stress ( Table 2 ). The shear force values of the pectoralis in the heat-stressed groups increased after 1 and 2 h of heat stress (0.01). After 3 h of heat stress, the shear force values gradually decreased (0.05) but were still higher than in the control group. No significant (0.05) differences were observed between the shear force values of the heat-stressed groups and the control group after 10 h of heat stress. The meat color lightness and yellowness values increased slightly with heat stress. A significant increase in the lightness value was observed after both 3 and 5 h of heat stress (0.01). An increase in the yellowness value was detected after 5 h of heat stress (0.05). However, compared with the control group, the redness value of the pectoralis in the heat-stressed groups decreased significantly (0.01) shortly after exposure to heat stress.

Circulating parameters, such as CK, GPT, and GOT levels, that are associated with cellular injury in broilers exposed to high temperature at an early age are shown in Table 1 . The levels of plasma CK and GPT gradually increased shortly after the birds were exposed to sudden heat stress, and the levels of circulating enzymes remained high until the heat treatment was terminated. Plasma CK levels increased (0.01) after 2 h of heat stress and reached a maximum (0.01) after 10 h. Plasma GPT levels increased after 3 h (<0.05), and reached a maximum after 10 h (<0.01). GOT levels were not statistical significant (>0.05). Compared with the control group, plasma insulin levels in the heat-stressed groups exhibited a sharp decline shortly after exposure to heat stress (0.01). Compared with the control group, plasma glucagon concentrations remained not significant (>0.05) from 1 h to 2 h of heat stress, then began to decrease gradually from 3 h to 5 h (0.05), followed by a further decline (0.01) to 10 h of heat stress.

DISCUSSION

P<0.01) upon exposure to heat stress; thereafter, the levels remained very low. These observations are consistent with previous reports (Itoh et al.

Insulin and glucagon are the most important hormones that control glucose metabolism. The data in Table 1 may indicate that the release of plasma insulin and glucagon was inhibited during the heat stress period. A significant reduction in chicken plasma insulin levels was observed immediately (0.01) upon exposure to heat stress; thereafter, the levels remained very low. These observations are consistent with previous reports (Itoh et al. 2001 ; Atkinson et al. 1981 ; Kurose et al. 1990 ) showing that decreased insulin levels during heat stress are the result of the inhibition of α-stimulation (simultaneous propranolol and epinephrine infusion). However we cannot ignore that, during heat stress, insulin and glucagon levels could be depleted or bound to receptors, thus making accurate analysis difficult.

P<0.05) and 10 h (P<0.01) of heat stress. The insulin decreased (P<0.01) along the time of heat stress. Glucagon levels began to decrease (P<0.05) following 5 h of heat stress and reached the lowest level after 10 h (P<0.01). In contrast, after 10 h of heat stress, insulin levels began to increase significantly (P<0.01) compared with the levels detected at 5 h. This may indicate the opposing effects of insulin and glucagon on glucose homeostasis during acute heat stress. Similar effects have been reported in heat-stressed sheep, suggesting that the insulin secretory response is inhibited by heat exposure (Itoh et al.

The levels of plasma glucagon decreased markedly after 5 h (0.05) and 10 h (0.01) of heat stress. The insulin decreased (0.01) along the time of heat stress. Glucagon levels began to decrease (0.05) following 5 h of heat stress and reached the lowest level after 10 h (0.01). In contrast, after 10 h of heat stress, insulin levels began to increase significantly (<0.01) compared with the levels detected at 5 h. This may indicate the opposing effects of insulin and glucagon on glucose homeostasis during acute heat stress. Similar effects have been reported in heat-stressed sheep, suggesting that the insulin secretory response is inhibited by heat exposure (Itoh et al. 2001 ). Considerable inconsistency, however, has been observed among different studies of the effects of heat exposure. In the present study, insulin exhibited a marked decline in the heat-stressed broilers. The sensitivity of insulin-dependent tissues declines as the secretion of insulin is depressed on exposure to high temperature (Oda et al. 1988 ; Kurose et al. 1990 ). Under such conditions, greater amounts of glucose are available to insulin-independent tissues, such as the cerebrum and medulla, thus fulfilling the requirements of important organs (Jones et al. 2012 ). Changes in insulin and glucagon secretion from the pancreas have previously been reported in broilers (Bao et al. 2004 ; Dridi et al. 2008 ) and sheep (Achmadi et al. 1993 ). However, the mechanism underlying the decrease in plasma glucagon levels observed in heat-stressed broilers requires further investigation.

Exposure to high ambient temperature markedly increased plasma CK and GPT levels in the broilers after different periods of heat stress, although heat stress had no significant effect on GOT levels. In the present study, plasma CK and GPT levels increased significantly as the duration of heat stress was increased. Increased plasma CK levels are indicative of skeletal muscle damage and are a consequence of a disruption in the function and permeability of the muscle cell membrane (Mitchell and Sandercock 1995 ; Brancaccio et al. 2007 ).

2+-mediated alterations in muscle membrane integrity (Mitchell and Sandercock 2+-mediated changes in muscle membrane permeability are caused by hyperthermia is not possible, we can conclude that the damage to the muscle becomes more severe as the duration of heat stress increases. Previous studies demonstrated that CK loss occurs as a result of the dysregulation of muscle intracellular Ca2+ homeostasis (Sandercock and Mitchell

Hyperthermia-associated myopathy is also characterized by an increase in the plasma activity of the skeletal-muscle-derived isoenzyme CK, reflecting Ca-mediated alterations in muscle membrane integrity (Mitchell and Sandercock 1997 ). Although, based on the results of this study, asserting that Ca-mediated changes in muscle membrane permeability are caused by hyperthermia is not possible, we can conclude that the damage to the muscle becomes more severe as the duration of heat stress increases. Previous studies demonstrated that CK loss occurs as a result of the dysregulation of muscle intracellular Cahomeostasis (Sandercock and Mitchell 1998 1999 ). Our results reveal that the pH value of the chicken pectoralis significantly decreased during the heat stress period ( Table 2 ). Based on the data presented in Table 3 , plasma CK and pectoralis pH were negatively correlated. Similar increases in plasma CK levels have been reported under hyperthermia-associated stress conditions in pigs and shown to be associated with detrimental effects on meat quality (Shibata 1996 ). The concentrations of plasma CK and GPT can be considered parameters for assessing the stress condition of broilers.

P<0.01) by heat stress, reaching its lowest value after 5 h. It can be speculated that this effect is a consequence of an increased rate of muscle glycolytic metabolism immediately post-mortem. Furthermore, changes in pHi are related to changes in deep-body temperature, acid/base state, and degree of muscle damage (Froning et al. r) for the relationship between pHi and pHu was 0.560, implying a significant correlation between these two parameters (

The effects of acute heat stress on post-mortem meat quality parameters are shown in Table 2 . The present study clearly shows that acute heat stress markedly changed blood chemistry, indicating skeletal muscle cell injury during heat stress. These ante-mortem pathophysiological phenomena appear to be associated with changes in post-mortem chicken pectoralis and meat characteristics. The pectoralis pHi value decreased significantly (<0.01) by heat stress, reaching its lowest value after 5 h. It can be speculated that this effect is a consequence of an increased rate of muscle glycolytic metabolism immediately post-mortem. Furthermore, changes in pHi are related to changes in deep-body temperature, acid/base state, and degree of muscle damage (Froning et al. 1978 ). Measurements of breast meat pHu at 24 h post-chilling at 4°C revealed a similar tendency in pHi ( Table 2 ). The Pearson correlation coefficient () for the relationship between pHi and pHu was 0.560, implying a significant correlation between these two parameters ( Table 3 ). The pHu of heat-stressed lower pectoralis may have led to changes in WHC, shear force value, and meat color (Van Laack et al. 2000 ).

The water-holding capacity of fresh meat (the ability to retain inherent water) is an important property of fresh meat, as it affects both the yield and the quality of the end-product. This parameter is reflected by cooking loss and expressible moisture. Muscle water content can be divided into three forms: bound water (5%), immobilized water (80%) and free water (15%). With the exception of the bound form, water can be driven off by conventional heating or affected by lower pH, which can contribute to myofibril shrinkage and consequential water loss (Honikel 1998 ). The cooking loss and expressible moisture of chicken pectoralis in this study represented the immobilized and free water content. Our results clearly demonstrated identical tendencies in the cooking loss and expressible moisture of heat-stressed broiler breast meat ( Table 2 ). Numerous reviews have discussed the factors that influence WHC (Honikel 2004 ). The main determinants of the WHC of meat are pH and protein denaturation (Offer and Knight 1988 ; Van Laack et al. 2000 ; Yusop et al. 2010 ). The pH and WHC of pectoralis were significantly negatively correlated ( Table 3 ). The decrease in pHu may explain the relatively low WHC of heat-stressed broiler breast meat. The pH and WHC of the breast meat were shown to have similar inflexions in the 5-h heat-stressed broilers ( Table 2 ).

P>0.05) compared with the control group. One of the most important factors affecting meat tenderness is the action of proteases, such as calpains, which exhibit optimal activity at approximately neutral pH. In the present study, the pH values of meat were decreased before 5 h of heat stress, indicating reduced calpain activity (Huff Lonergan and Lonergan

In the present study, the shear force value and pHu were negatively correlated ( Table 3 ). The higher cooking loss and expressible moisture indicated the lower WHC. Also, the decrease in pHu may explain the relatively low WHC of heat-stressed broiler pectoralis. As the period of heat stress was increased, the shear force values of chicken pectoralis in the heat-stressed groups clearly increased and then decreased slightly after 3 and 5 h of heat stress ( Table 2 ). After 10 h of heat stress, no significant difference was observed in meat tenderness (0.05) compared with the control group. One of the most important factors affecting meat tenderness is the action of proteases, such as calpains, which exhibit optimal activity at approximately neutral pH. In the present study, the pH values of meat were decreased before 5 h of heat stress, indicating reduced calpain activity (Huff Lonergan and Lonergan 1999 , 2010) and leading to the reduction of WHC (Morrison et al. 1998 ). At 10 h of heat stress, pH values were increased and closer to the optimal pH for calpains (Kendall et al. 1993 ; Pomponio et al. 2010 ). Furthermore, WHC values increased to levels higher than those detected at 5 h of heat stress. Therefore, low tissue pH would depress the activity of these proteases, leading to an increased shear force value from the beginning of heat stress. After 3 h of heat stress, the observed reduction in the shear force value may reflect acclimatization of the surviving broilers to the high temperature. This is consistent with previous reports that indicate that the early (1–2 h) effects of heat stress are the most detrimental (Yu et al. 2008 ). This adaptation may account for the improvement in shear force values of broiler breast muscle at later time-points during the heat stress period compared with those observed at 1 to 2 h. However, the underlying mechanism of this effect remains to be elucidated.

Interestingly, in the present study, the lowest pH and highest shear force value did not coincide at the same time-point in the heat stress period ( Table 2 ). This suggests that pH is not the only factor that influences the shear force value as the duration of heat stress is increased. The post-mortem temperature of muscle, for example, has been shown to be an important factor that influences tenderness (Marsh et al. 1981 ).

lightness and a lower yellowness compared with controls. At 10 h, the pHu and WHC values increased compared with those observed after 5 h of heat stress, while the lightness value decreased to normal levels. In contrast, lightness was found to correlate negatively with pH. This finding is consistent with previous reports (Yang and Chen redness values is dependent on the consistently low pH values, which contribute to the unusual redox reaction of myoglobin and hemoglobin (Froning et al. yellowness values of the breast fillets in different heat-stressed broilers were not significantly different from the controls. These observations may be caused by the type of muscle as the lightness and redness values are more important and sensitive than yellowness values in white muscle (Barbut

The color of meat is an important visible parameter that reflects meat quality. The lightness, redness, and yellowness color values of the breast fillets are presented in Table 2 . When the pH of meat is greater than the isoelectric point of the myofibrillar proteins in the meat, water molecules are tightly bound, causing more light to be absorbed by the muscle, and the meat appears darker in color (Cornforth 1994 ). In our preliminary study, the breast muscle of broilers exposed to high temperature exhibited a higherand a lowercompared with controls. At 10 h, the pHu and WHC values increased compared with those observed after 5 h of heat stress, while thevalue decreased to normal levels. In contrast,was found to correlate negatively with pH. This finding is consistent with previous reports (Yang and Chen 1993 ; Allen et al. 1997 ; Aksit et al. 2006 ; Wang et al. 2010 ). The continuous decrease ofvalues is dependent on the consistently low pH values, which contribute to the unusual redox reaction of myoglobin and hemoglobin (Froning et al. 1978 ). However, thevalues of the breast fillets in different heat-stressed broilers were not significantly different from the controls. These observations may be caused by the type of muscle as theandvalues are more important and sensitive thanvalues in white muscle (Barbut 1997 ).

The results obtained from this study indicate that acute ante-mortem heat stress in AA broilers has a marked influence on blood parameters and breast meat quality attributes. Plasma CK and GPT concentrations gradually increased shortly after the birds were exposed to sudden heat stress, and enzyme levels were maintained at high levels until the termination of heat treatment. Our results demonstrate that elevation of plasma CK and GPT levels are concomitant with increases in the duration of heat stress, and are linked to cellular damage. This may suggest that heat stress causes cell damage in the muscle, which causes a decline in meat quality. The values of both pHi and pHu in chicken pectoralis were found to be lower in heat-stressed chickens compared with normal chickens (P<0.01). However, the cooking loss and expressible moisture of pectoralis in the heat-stressed lines gradually increased. Broilers exposed to high temperature had a higher shear value, L * value, and b * value. However, the a * value of the pectoralis in the heat-stressed groups significantly decreased (P<0.01) shortly after heat stress exposure. The pH value clearly decreased, concomitant with the increase in plasma CK activity after heat stress. Cooking loss and expressible moisture, however, exhibited a positive correlation. The increases in cooking loss, expressible moisture, and shear force value may be consequences of the decrease in pH value. Our data indicate that preslaughter exposure of broiler chickens to acute heat stress should be avoided to reduce alterations in muscle metabolism and membrane integrity and, consequently, undesirable meat characteristics.

In conclusion, acute heat stress is detrimental to the meat quality of broiler chickens. It is important to control the animal environment to minimize heat stress and to reduce the mortality associated with it. Feed additives, vitamins, nutritional and/or management tools should be investigated in order to address problems associated with heat stress and ameliorate its effect. Furthermore, the mechanism responsible for heat-induced sudden death also requires in-depth investigations. A clear understanding of the mechanisms underlying these processes will contribute to the determination of prevention strategies and the avoidance of the associated economic losses.