Heat stress is of major concern for poultry, especially in the hot regions of the world because of the resulting poor growth performance, immunosuppression, and high mortality. To assess superoxide (O2*-) production in mitochondria isolated from skeletal muscle of chickens (n = 4 to 8) exposed to acute heat stress, electron spin resonance (ESR) spectroscopy using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap agent and lucigenin-derived chemiluminescence (LDCL) method were applied. ESR spectra of suspensions containing mitochondria from control and acute heat-treated meat-type chickens showed similar hyperfine coupling constants (aN = 1.44 mT, aHbeta = 0.12 mT, and aHbeta = 0.11 mT) to those of DMPO-O2*- adducts observed in a hypoxanthine-xanthine oxidase system. Heat exposure resulted in enhancement of the DMPO-O2*- signal. The results using LDCL showed significantly enhanced superoxide production in heat stress-treated skeletal muscle mitochondria of meat-type chickens, whereas no such increase was observed in laying chickens. The enhancement of superoxide production in the former case was associated with heat-induced increments in rectal and muscle temperatures, leading to significant body weight loss. In contrast, the latter case showed no increase in temperatures, although there was a slight decrease in body weight gain. Percentage increases of superoxide production in the presence of carboxyatractylate, a specific inhibitor of adenine nucleotide translocator (ANT), were the same for skeletal muscle mitochondria from meat- and laying-type chickens from the control or heat stress-treated group. This finding suggests the irrelevance of ANT in the regulation of reactive oxygen species flux under heat stress conditions. The study provides the first evidence of superoxide anion production in the skeletal muscle mitochondria of meat-type chickens in response to acute heat stress.

Broiler chickens are highly sensitive to high ambient temperatures due to their feathers, lack of skin sweat glands, and high productivity. Heat stress (HS) is a major concern for the poultry industry because it negatively affects growth as well as immune functions, which increase the potential risk of infectious disease outbreaks. Therefore, it is vital to elucidate HS's effect on the avian immune system, especially considering the global rise in average surface temperature. Our study identified a series of immunological disorders in heat-stressed broiler chickens. We exposed 22-day-old broiler chickens to a continuous HS condition (34.5 ± 0.5°C) for 14 days and immunized them with a prototype bovine serum albumin (BSA) antigen. The plasma and lymphoid tissues (thymus, bursa of Fabricius, and spleen) were harvested at the end of the experiments to investigate the induction of BSA-specific immune responses. Our results revealed that plasma titers of immunoglobulin (Ig)Y, IgM, and IgA antibodies specific for BSA were lower than those of thermoneutral chickens immunized with BSA. Furthermore, the spleens of the heat-stressed broiler chickens displayed severe depression of Bu1⁺ B cells and CD3⁺ T cells, including CD4⁺ T cells and CD8⁺ T cells, and lacked a fully developed germinal center (GC), which is crucial for B cell proliferation. These immunological abnormalities might be associated with severe depression of CD4⁻CD8⁻ or CD4⁺CD8⁺ cells, which are precursors of either helper or killer T cells in the thymus and Bu1⁺ B cells in the bursa of Fabricius. Importantly, HS severely damaged the morphology of the thymic cortex and bursal follicles, where functional maturation of T and B cells occur. These results indicate that HS causes multiple immune abnormalities in broiler chickens by impairing the developmental process and functional maturation of T and B cells in both primary and secondary lymphoid tissues.

Mitochondria have long been known to play a critical role in maintaining bioenergetic status under physiological conditions. Our previous studies have shown that acute heat stress increases production of mitochondrial reactive oxygen species (ROS) in the skeletal muscle of chickens. This increased ROS production may lead to nonspecific modification of lipids and proteins, which may then result in bioenergetic dysfunctions. If increased mitochondrial ROS production in heat-stressed chickens causes oxidative damage, changes in mitochondrial peroxidized lipids and oxidatively modified proteins can be detected. To study this, 3-week-old male broiler chickens (n=4-8) were exposed to acute heat stress (34°C for 18h) while control chickens were kept at thermoneutral condition (25°C). Skeletal muscle subsarcolemmal mitochondria were isolated and used to study mitochondrial malondialdehyde (MDA) and protein carbonyl groups. Mitochondrial thiobarbituric acid reactive substances (TBARS) formation was measured colorimetrically with 0.01% butylated hydroxytoluene (BHT) and expressed as MDA equivalent. To detect oxidation-sensitive mitochondrial proteins, polypeptides resolved by two-dimensional (2D) electrophoresis were immunostained with DNP-specific antibodies for carbonylated proteins using Western blotting. In heat-stressed chickens, mitochondrial MDA was 2.7-fold higher, and 82 mitochondrial proteins were oxidized when compared to that of control chickens. These results suggest that in heat-stressed chickens, increased mitochondrial ROS production leads to oxidative damage to mitochondrial lipids and proteins.

Acute heat stress (34°C for 18 h) resulted in increased levels of reactive oxygen species (ROS) in mitochondria isolated from the skeletal muscle of broilers. This occurred when glutamate-requiring complexes I, III, and IV of the electron transport chain or succinate-requiring complexes II, III and IV were used as the substrate. This result confirms our previous observation that exposure of broilers to 34°C for 18 h results in increased superoxide production in skeletal (pectoralis) muscle, and extends this finding by showing that substrate-independent ROS generation occurs during the heat stress period. When broilers were exposed to heat stress, the levels of avian uncoupling protein (avUCP) mRNA in skeletal muscle were significantly decreased, to 28% of the levels found in untreated controls. This was accompanied by a significant decrease in the levels of the avUCP protein, to 37% of control levels. In contrast, avian adenine nucleotide translocator mRNA levels were not affected by exposure to heat stress. This finding is consistent with previous studies which showed that the increases in superoxide production that are observed in the presence of carboxyatractylate, a specific inhibitor of adenine nucleotide translocator, were the same for skeletal muscle mitochondria from both control and heat-stressed chickens. Taken together, these results suggest that acute heat stress stimulates mitochondrial superoxide production in broiler skeletal muscle, possibly via downregulation of avUCP. The present study provides the first evidence that synthesis of avUCP protein is downregulated in heat-stressed broilers.

1. The present study was undertaken to determine the effects of dietary spirulina on growth performance and pigmentation in the muscle of growing broiler chickens and to examine the possibility that zeaxanthin in spirulina may affect yellow colour development in the meat. 2. Twenty-four, 21-d-old, male broiler chicks were fed an experimental diet containing spirulina at 0, 40, or 80 g/ kg for 16 d. No significant differences among treatments were observed in body weights, nor weights or yields (as a percentage of body weight) for any of the selected traits, including liver, abdominal fat, kidney and Pectoralis profundus. 3. Spectrocolourimetric analyses revealed that the redness of Pectoralis superficialis, profundus and Sartorius muscles reached a maximum in chicks fed the 40 g/kg spirulina diet, while the yellowness of all fillets, including the Semitendinosus muscle, increased in a sub-linear fashion with increased spirulina in the diet. The overall correlation between the yellowness and zeaxanthin content in the Pectoralis muscle was significant. 4. This study provides the first conclusive evidence that dietary spirulina influences both the yellowness and redness of broiler flesh and that the increments in yellowness with dietary spirulina content may possibly be reflected in the common yellow pigment related to the accumulation of zeaxanthin within the flesh.