Using Recombinant Superoxide Dismutase to Control Oxidative Stress in the Gastrointestinal Tract of Cyclic Heat-Stressed Pigs.

Climate change is associated with an increased frequency and intensity of heat waves, posing a threat of heat stress to pig production. Heat stress compromises the efficiency of pig production partly due to causing oxidative stress, intestinal dysfunction, and inflammatory responses. Superoxide dismutase is an antioxidant enzyme reported to reduce oxidative stress and inflammation. Therefore, this experiment aimed to investigate whether recombinant superoxide dismutase (rSOD) could ameliorate oxidative stress and inflammatory responses in heat-stressed grower pigs. Sixty-four female pigs (Large White × Landrace, 27.8 ± 1.65 kg, mean ± SD) were randomly allocated to a control diet (standard grower feed, CON) or the control diet supplemented with 50 IU recombinant superoxide dismutase (rSOD) for 14 days. After acclimation to the diet, pigs were then housed under thermoneutral (TN, 20 °C, 35-50% relative humidity) or cyclic heat stress conditions (CHS, at 35 °C: 9 a.m. to 5 p.m. and 28 °C: 5 p.m. to 9 a.m., 35-50% relative humidity) for 3 days. Heat stress increased respiration rate (RR), skin and rectal temperature (RR and RT) (p < 0.001 for all), and reduced plasma thyroid hormone concentration (p < 0.001). The amount of oxidized glutathione (GSH:GSSG) was increased in the jejunum and ileum of CHS pigs. In the jejunum, rSOD also increased the amount of oxidized glutathione in both TN and CHS pigs, without any change in endogenous SOD activity. In the ileum, rSOD prevented increases in oxidized glutathione formation in the CHS pigs only. Taken together, this may reflect increased oxidative stress in both the jejunum and ileum in CHS pigs. Alternatively, rSOD increased the conversion of reduced to oxidized glutathione independently of CHS, possibly reflecting an increased overall SOD activity due to the addition of exogenous SOD. In conclusion, the use of in-feed SOD enzymes at a dose of 50 IU/kg may be a useful strategy for preventing oxidative stress in pigs.

G protein-coupled receptor interactions and modification of signalling involving the ghrelin receptor, GHSR1a.

The growth hormone secretagogue receptor 1a (GHSR1a) is intriguing because of its potential as a therapeutic target and its diverse molecular interactions. Initial studies of the receptor focused on the potential therapeutic ability for growth hormone (GH) release to reduce wasting in aging individuals, as well as food intake regulation for treatment of cachexia. Known roles of GHSR1a now extend to regulation of neurogenesis, learning and memory, gastrointestinal motility, glucose/lipid metabolism, the cardiovascular system, neuronal protection, motivational salience, and hedonic feeding. Ghrelin, the endogenous agonist of GHSR1a, is primarily located in the stomach and is absent from the central nervous system (CNS), including the spinal cord. However, ghrelin in the circulation does have access to a small number of CNS sites, including the arcuate nucleus, which is important in feeding control. At some sites, such as at somatotrophs, GHSR1a has high constitutive activity. Typically, ghrelin-dependent and constitutive GHSR1a activation occurs via Gαq/11 pathways. In vitro and in vivo data suggest that GHSR1a heterodimerises with multiple G protein-coupled receptors (GPCRs), including dopamine D1 and D2, serotonin 2C, orexin, oxytocin and melanocortin 3 receptors (MCR3), as well as the MCR3 accessory protein, MRAP2, providing possible mechanisms for its many physiological effects. In all cases, the receptor interaction changes downstream signalling and the responses to receptor agonists. This review discusses the signalling mechanisms of GHSR1a alone and in combination with other GPCRs, and explores the physiological consequences of GHSR1a coupling with other GPCRs.

Betaine and Isoquinoline Alkaloids Protect against Heat Stress and Colonic Permeability in Growing Pigs.

Heat stress (HS) compromises productivity of pork production, in part as a result of increased oxidative stress and inflammatory responses, particularly within the gastrointestinal tract. This study aimed to investigate whether plant-derived betaine and isoquinoline alkaloids could ameliorate HS in pigs. Fifty female Large White × Landrace grower pigs, which were acclimated to control (CON), control plus betaine (BET), or control plus isoquinoline alkaloids (IQA) diets for 14 days were then exposed to heat stress or thermoneutral condition. Both BET and IQA partially ameliorated increases in respiration rate (p = 0.013) and rectal temperature (p = 0.001) associated with HS conditions. Heat stress increased salivary cortisol concentrations and reduced plasma creatinine, lactate, and thyroid hormone concentrations. Heat stress increased colon FD4 permeability, which was reduced by IQA (p = 0.030). Heat stress increased inflammation in the jejunum and ileum, as indicated by elevated interleukin-1ß (p = 0.022) in the jejunum and interleukin-1ß (p = 0.004) and interleukin-8 (p = 0.001) in the ileum. No differences in plasma total antioxidant capacity (TAC) were observed with HS, but betaine increased plasma TAC compared to IQA. Dietary BET increased betaine concentrations in the jejunum, ileum (p < 0.001 for both), plasma, liver, kidney (p < 0.010 for all), urine (p = 0.002) and tended to be higher in muscle (p = 0.084). Betaine concentration was not influenced by HS, but it tended to be higher in plasma and accumulated in the liver. These data suggest that betaine and isoquinoline alkaloids supplementation ameliorated consequences of heat stress in grower pigs and protected against HS induced increases in colonic permeability.

Dietary Betaine Improves Intestinal Barrier Function and Ameliorates the Impact of Heat Stress in Multiple Vital Organs as Measured by Evans Blue Dye in Broiler Chickens.

In a 2 × 2 factorial design, 60 male Ross-308 broilers were fed either a control or 1 g/kg betaine diet and housed under thermoneutral (TN) or heat stress (HS) conditions. Broilers were acclimated to diets for 1 week under TN (25 °C), then either kept at TN or HS, where the temperature increased 8 h/day at 33 °C and 16 h/day at 25 °C for up to 10 days. Respiration rate (RR) was measured at four time points, and on each of 1, 2, 3, 7 and 10 days of HS, 12 broilers were injected with 0.5 mg/kg of Evans Blue Dye (EBD) solution to quantify regional changes in tissue damage. Betaine was quantified in tissues, and ileal damage was assessed via morphometry and transepithelial resistance (TER). Heat stress elevated RR (p < 0.001) and resulted in reduced villous height (p = 0.009) and TER (p < 0.001), while dietary betaine lowered RR during HS (p < 0.001), increased betaine distribution into tissues, and improved ileal villous height (p < 0.001) and TER (p = 0.006). Heat stress increased EBD in the muscle and kidney of chickens fed the control diet but not in those receiving betaine. Overall, these data indicate that supplemented betaine is distributed to vital organs and the gastrointestinal tract, where it is associated with improved tolerance of HS. Furthermore, EBD markers help reveal the effects of HS on organs dysfunction.

Betaine and Antioxidants Improve Growth Performance, Breast Muscle Development and Ameliorate Thermoregulatory Responses to Cyclic Heat Exposure in Broiler Chickens.

Heat stress (HS) is an environmental stressor challenging poultry production and requires a strategy to cope with it. A total of 288-day-old male broiler chicks were fed with one of the following diets: basal diet, basal with betaine (BET), or with selenium and vitamin E (AOX), or with a combination of BET and AOX, under thermoneutral and cyclic HS. Results showed that HS reduced average daily feed intake (ADFI) (p = 0.01) and average daily gain (ADG) (p < 0.001), and impaired feed conversion ratio (FCR) (p = 0.03) during rearing period (0⁻42 day). BET increased ADG (p = 0.001) and decreased FCR (p = 0.02), whereas AOX had no effects. Breast muscle weight was decreased by HS (p < 0.001) and increased by BET (p < 0.001). Rectal temperature was increased by HS (p < 0.001) and improved by BET overall. Respiration rate was increased by HS (p < 0.001), but BET decreased it during HS (p = 0.04). Jejunum transepithelial resistance was reduced by HS and had no effect on permeability whereas BET increased jejunum permeability (p = 0.013). Overall, the reductions in ADG of broiler chickens during HS were ameliorated by supplementation with BET, with much of the increase in ADG being breast muscle.

Selenium-enriched Agaricus bisporus mushroom protects against increase in gut permeability ex vivo and up-regulates glutathione peroxidase 1 and 2 in hyperthermally-induced oxidative stress in rats.

Dietary effects of organic Se supplementation in the form of Se-enriched Agaricus bisporus mushroom on ileal mucosal permeability and antioxidant selenoenzymes status in heat induced oxidative stress in rats were evaluated. Acute heat stress (40 °C, 21% relative humidity, 90 min exposure) increased ileum baseline short circuit current (Isc; 2.40-fold) and epithelial conductance (Ge; 2.74-fold). Dietary supplementation with Se-enriched A. bisporus (1 µg Se/g feed) reduced (p < 0.05) ileum Isc and Ge during heat stress to 1.74 and 1.91 fold, respectively, indicating protection from heat stress-induced mucosal permeability increase. The expression of ileum glutathione peroxidase (GPx-) 1 and 2 mRNAs were up-regulated (p < 0.05) by 1.90 and 1.87-fold, respectively, for non-heat stress rats on the Se-enriched diet relative to the control. The interplay between heat stress and dietary Se is complex. For rats on the control diet, heat stress alone increased ileum expression of GPx-1 (2.33-fold) and GPx-2 (2.23-fold) relative to thermoneutral conditions. For rats on the Se-enriched diet, heat stress increased (p < 0.05) GPx-1 expression only. Rats on Se-enriched + α-tocopherol diet exhibited increased expression of both genes (p < 0.05). Thus, dietary Se-enriched A. bisporus protected against increase in ileum permeability and up-regulated GPx-1 and GPx-2 expression, selenoenzymes relevant to mitigating oxidative stress.

Effects of protein deprivation and re-feeding on P2X2 receptors in enteric neurons.

AIM: To investigate the effects of malnutrition and re-feeding on the P2X(2) receptor, nitric oxide synthase (NOS), calretinin, calbindin and choline acetyltransferase (ChAT) in neurons of the rat ileum. METHODS: We analyzed the co-localization, numbers and sizes of P2X(2)-expressing neurons in relation to NOS-immunoreactive (IR), calbindin-IR, ChAT-IR, and calretinin-IR neurons of the myenteric and submucosal plexus. The experimental groups consisted of: (1) rats maintained on normal feed throughout pregnancy until 42 d post-parturition (N); (2) rats deprived of protein throughout pregnancy and 42 d post-parturition (D); and (3) rats undernourished for 21 d post-parturition and then given a protein diet from days 22 to 42 (DR). The myenteric and submucosal plexuses were evaluated by double labeling by immunohistochemical methods for P2X(2) receptor, NOS, ChAT, calbindin and calretinin. RESULTS: We found similar P2X(2) receptor immunoreactivity in the cytoplasm and surface membranes of myenteric and submucosal neurons from the N, D and DR groups. Double labeling of the myenteric plexus demonstrated that approximately 100% of NOS-IR, calbindin-IR, calretinin-IR and ChAT-IR neurons in all groups also expressed the P2X(2) receptor. In the submucosal plexus, the calretinin-IR, ChAT-IR and calbindin-IR neurons were nearly all immunoreactive for the P2X(2) receptor. In the myenteric plexus, there was a 19% increase in numbers per cm(2) for P2X(2) receptor-IR neurons, 64% for NOS-IR, 84% for calretinin-IR and 26% for ChAT-IR neurons in the D group. The spatial density of calbindin-IR neurons, however, did not differ among the three groups. The submucosal neuronal density increased for calbindin-IR, calretinin-IR and ChAT-IR neurons. The average size of neurons in the myenteric plexus neurons in the D group was less than that in the controls and, in the re-fed rats; there was a 34% reduction in size only for the calretinin-IR neurons. CONCLUSION: This work demonstrates that expression of the P2X(2) receptor is present in inhibitory, intrinsic primary afferent, cholinergic secretomotor and vasomotor neurons. Undernutrition affected P2X(2) receptor expression in the submucosal plexus, and neuronal and size. These changes were rescued in the re-fed rats.