INTRODUCTION
Cold stress is a great challenge for broilers during winter transportation. It is estimated that 20 million poultry deaths and an economic loss of 100 million can be attributed to the winter conditions and low temperatures in China [
1]. In a low temperature, the metabolic rate of fast-growing broilers rises for heat production to maintain normal body temperature, leading to excess nutrient oxidation and oxygen deficiency [
2]. Consequently, cardiac output increases to supply more oxygen for metabolism, which results in pathological alterations such as classic hematological changes and heart hypertrophy [
3]. Long-standing pathologic hypertrophy will contribute to heart disorder and eventually increases the mortality and morbidity of chickens [
4]. Of note, it has been well documented that exposure to low temperature induced the elevation of oxidative metabolism and heat production, accompanied by elevated reactive oxygen species (ROS) production and the oxidative stress in heart and liver [
5]. In turn, oxidative damage further aggravates heart and liver dysfunction, which weakens the chickens. Additionally, cold stress has been also reported to impair the energy status by reducing cardiac energetic reserve, hepatic fatty acid oxidation and respiratory capacity [
6].
To minimize the impairment caused by cold temperature, the techniques of nutritional manipulation are increasingly developed in broiler production, for example, dietary supplementation with vitamin C [
7], copper-methionine [
8], arginine and guanidinoacetic acid [
2]. N-acetylcysteine (NAC) is a precursor of L-cysteine, which is utilized for the synthesis of reduced glutathione (GSH). A large body of evidence showed that NAC exhibited direct and indirect antioxidant properties via its free thiol group and the increase of cellular GSH concentration, respectively [
9]. NAC has been in clinical practice for several decades and used for the treatment of cardiac injury, pulmonary disease, and cancer [
10]. Intriguingly, in addition to its antioxidant actions, NAC may act as a vasodilator by facilitating the production and action of nitric oxide [
11], implying that NAC plays a pivotal role in attenuating the constriction of blood vessels induced by cold temperature. Additionally, we have reported dietary NAC alleviated liver injury in a porcine model by improving the antioxidative capacity and energy metabolism [
12]. Similarly, NAC also alleviates the toxic effects of aflatoxin in broilers by improving antioxidant capacity and energy metabolism [
13]. Therefore, we hypothesized that NAC might benefit the hepatic and cardiac function of cold-stressed boilers by modulating the energy and antioxidant status. The present study was carried out to investigate the effects of NAC on the antioxidant capacity and energy status of liver and heart in broilers exposed to the low temperature, thereby providing the theoretical basis for the NAC application in poultry production.
DISCUSSION
Broilers are sensitive to temperature variations and their metabolic rate will rise to adapt to colder conditions [
18], and thereby leading to the growth inhibition and even death [
3]. Low ambient temperature is reported to induce hypoxia and ascites in broilers due to the increase of oxygen demands for both fast growth and heat production [
3]. Several managements including nutritional and medicinal strategies are taken to minimize the loss in broilers due to the exposure to cold stress. Herein, we determined whether dietary NAC could improve hepatic and cardiac redox status and energy status in broilers under the low temperature based on the beneficial effects of NAC on heart, liver and intestinal of animals [
11,
12].
In the present study, the exposure to low temperature greatly impaired the growth performance of broilers. This was unsurprising, because heat production increased to maintain the normal body temperature of cold-stressed broilers and more nutrients were used for heat production [
3]. As expected, cold stress increased the relative weights of heart and liver since low-temperature is an important inducer for hypertrophic growth of heart [
4]. Moreover, cold stress decreased the relative weights of both thymus and bursa, which were responsible for the generation of T and B lymphocytes, respectively. It was reported that cold stress could suppress the immune responses of chickens [
19]. Dietary NAC mitigated the decrease of bursa relative weight in cold-stressed broilers, which might further modulate the function of B lymphocytes that derived from bursa in chickens. In an
in vitro experiment, NAC regulated the homeostasis of CD40-activated B lymphocytes isolated from human peripheral blood and showed immunomodulatory function with antioxidant-independent properties [
20]. Therefore, NAC might play an immunomodulatory role in cold-stressed birds.
There are several indicators associated with hepatic diseases, such as the activities of ALT, AST, and ALP in blood [
21]. Of note, blood ALP is related to hepatic disease caused by intra or extra hepatic cholestatis and some destruction of hepatic cell membrane [
21]. Therefore, cold stress may result in liver dysfunction by observing that the activity of plasma ALP and relative weight of liver was increased in broilers exposed to low temperature in the present study. However, dietary NAC reduced the activity of plasma ALP in broilers under both thermoneutral and cold conditions, indicating that NAC could mitigate the liver injury. Additionally, the plasma TP and BUN levels were not affected by both treatments (diet and temperature), suggesting the protein metabolism was not influenced. Similar work in poultry is limited. However, in mammals, NAC was demonstrated to decrease BUN and improve kidney function through regulation of ammonia and nitrogen metabolism [
22,
23].
As mentioned above, cold stress could induce oxygen de ficiency (hypoxia), which would prevent the use of branched chain amino acids, including valine, leucine and isoleucine, in the mitochondrial electron transfer system of muscle [
24]. Therefore, in the current study, the increased concentrations of valine and isoleucine in plasma might be due to the oxygen deficiency in muscle induced by low ambient temperature, which needs further investigation. Unlike the finding of Muratsubaki and Yamaki [
24] that plasma lysine and methionine were not significantly affected by hypoxia, we found both were increased. These two amino acids might be modulated in a hypoxia-independent way under cold stress. Furthermore, cold stress reduced the levels of alanine, glutamine, proline, hydroxyproline, serine, and tryptophan in the plasma. This might be attributed to the liver injury (as mentioned above) and compromised intestinal integrity induced by cold stress [
19], which decreased the availability of these amino acids. As ATP was required for proline synthesis [
24], the reduced contents of ATP in heart and liver might also have contributed to the decrease of plasma proline level in broilers under cold condition. Additionally, NAC is a precursor of L-cysteine and undergoes extensive hepatic metabolism, resulting in increased levels of plasma cysteine, cystine, and GSH [
9], which were consistent with the results of present study.
Cold stress as well as the fast growth augmented the met abolic rate of chickens, giving rise to enhanced oxidative metabolism, lipid peroxidation, and ROS production [
25]. It is well known that ROS and related peroxides induced by cold stress lead to liver and heart injury. However, organisms can detoxify ROS using defense mechanisms (antioxidative enzymes) such as T-SOD, CAT, and GSH-Px [
13]. These antioxidant enzymes cooperatively convert ROS into oxygen and water. In the current study, birds under cold stress exhibited the elevation of cardiac MDA content and reduction of GSH-Px, CAT, and T-SOD activities, indicating that low temperature resulted in an oxidative status. Meanwhile, cold stress induced the down-regulation of hepatic and cardiac HIF-1α as well as cardiac HMOX. Our results were in good agreement with the study of Osselaere et al [
26], who also observed the down-regulation of hepatic HIF-1α and HMOX mRNA abundance in birds challenged with deoxynivalenol. HIF-1α is a core transcription factor regulating oxygen homeostasis and can activate the expression of many hypoxic reactive genes [
27], while HMOX is associated with the protection against hepatocyte death [
28]. It was stated that the up-regulation of HIF-1α usually happened in the first hours of oxygen deficiency and returned then to the basal level. In addition, cold stress also induced a down-regulation of cardiac XOR, an enzyme related to the synthesis of ROS [
29]. The reasons for the down-regulation of XOR by cold stress are not clear. As an antioxidant, NAC enhanced the activity of antioxidative enzyme such as hepatic T-SOD, CAT, and GSH-Px, and reduced hepatic MDA level of cold-stressed broilers. The underlying mechanism whereby NAC exerted antioxidative function might be directly and indirectly associated with oxidants as reported by Yi et al [
13]. Furthermore, NAC decreased the HIF-1α expression both in liver and heart but increased the HMOX and XOR mRNA levels in the heart of cold-stressed broilers, suggesting that NAC may regulate antioxidative capacity at a transcriptional level.
Except the elevations of triiodothyronine and leptin, cold stress increased the expression of uncoupling proteins, which uncoupled the respiration from ATP production to enhance body heat production and finally caused the reduction of ATP production [
30]. Moreover, sustained hypoxia during cold exposure could result in a loss of cardiac energetic reserve, hepatic fatty acid oxidation and respiratory capacity [
6]. Therefore, the present study observed the decrease of ATP concentration in liver and heart of cold-stressed broilers. Besides, cold stress increased the cardiac AMP/ATP ratio and reduced the hepatic AEC, indicating the impaired energy metabolism of birds. Unfortunately, dietary NAC did not show the improvements in energy metabolism, which may due to the insufficient experimental period. Nevertheless, NAC inhibited up-regulation of cardiac and hepatic AMPK, which serves as an energy sensor and is up-regulated by the elevating ratio of AMP/ATP [
31]. ATP5B is a key enzyme in catalyzing the synthesis of ATP [
32], and the up-regulation of cardiac ATP5B in cold-stressed broilers might be a feedback regulatory effects due to the decrease of ATP level in the heart. Another important gene, PGC-1α, which plays an important role in mitochondrial biogenesis and adaptive thermogenesis [
33], was down-regulated in the liver of broilers with cold stress or NAC supplementation. The impaired liver function in cold-stressed birds might be responsible for the down-regulation of PGC-1α. However, the underlying mechanisms how dietary NAC decreased the hepatic PGC-1α expression need further investigation.
In conclusion, cold stress decreased the growth perfor mance, altered the plasma amino acids profile, induced the oxidative stress and hypertrophy in liver and heart, and impaired the hepatic and cardiac energy metabolism of broilers. Dietary supplementation with 0.1% NAC mitigated the oxidative stress by increasing the activities of antioxidant enzymes in the liver of cold-stressed broilers. The 0.1% NAC is recommended to use in the diets of cold-stressed birds.