Study On The Regulatory Effect Of RRR-α-Tocopherol Succinate On Immune Stress In Broilers

Three experiments were conducted to examine the effects of supplemental D-TOS on immune response, antioxidant capacity, and meat quality in AA broiler chicks (a fast-growing meat type with higher sensitivity to stress reacton), also, the regulation effects on the immune response under immunological stress, finally, the protective effect on the oxidative damage of intestinal mucosa with its apoptosis regulation, and on mucosal immunity response. The comparision with all-rac-a-tocopherol acetate (DL-TOA) and/or RRR-a-acetate (D-TOA) were evaluated throughout the experiments.Firstly, the effects of dietary D-TOS supplementation on the growth, immunity, antioxidant capacity and meat quality in broilers was preliminarily explored. The immunity was investigated from the aspects of cellular, humoral immunity and neuroendocrine. The effects of two esters of a-tocopherol, DL-TOA and D-TOS were compared.320 one-day-old commercial AA broilers were randomly allocated to 4 treatments, each of which had 8 pens of 10 chicks per pen. Birds in the control group were fed with basal diets supplemented with 30 mg/kg DL-TOA, and those in the experimental groups with D-TOS supplementation of 10,30,50 mg/kg (TOS1, TOS2, TOS3 group), respectively for 42 d. The cellular immune response in vitro was measured using a lymphocyte isolation and blastogenesis assay. Antioxidant enzymes, concentrations of a-TOH, glutathione (GSH) and malonaldehyde (MDA) were measured by colorimetry assay. Serum T3, T4 and TSH were measured by radioimmunoassay and hepatic intracellular reactive oxygen species (ROS) assay by DCHF-DA fluorescent probe marking method. The results showed that,(1) With respect to the performance and immunity, there was no significant difference (P>0.05) in the growth performance among the four treatments. Index of spleen in group TOS3 (21 d and 42 d) had significant improvement than the control group (P<0.05), and index of thymus (21 d) and bursa (42 d) had been enhanced in comparison with group TOS1 (P<0.05). Serum anti-new-castlediseasevirus (NDV) titers were improved significantly in group TOS2 and TOS3 at day of 22 and 28 (P<0.05), while group TOS3 had the higher anti-avain influenza virus (AIV) antibody titers than group TOS1 (P<0.05). Marked enhancement of splenic T and B lymphocyte proliferation occurred in group TOS3 as compared to the other groups. The concentrations of serum glutathione (GSH) was highest at 50 mg/kg at 42 d of age (P<0.05), and hepatic GSH was significantly higher at 30 and 50 mg/kg compared to the other groups. Enhancement was observed on the serum T3, T4 and TSH levels with supplemention of 30-50 mg/kg D-TOS.(2) Concerning the antioxidant capacity and meat quality, significant positive correlations existed between dietary supplemental D-TOS levels and plasma (R2=0.9831, P<0.01) or hepatic (R=0.9336, P<0.05) a-TOH concentrations, and a negative correlation with plasma (R2=-0.9487, P<0.05) or hepatic (R2=-0.9901, P=0.0518) MDA levels. In comparison with the control and TOS1 group,30～50 mg/kg dietary D-TOS supplementation resulted in an increase in the activities of serum T-SOD, GSH-Px, T-AOC and hepatic T-SOD, T-AOC to some extent (P<0.05 orP<0.01). Furthermore, the level of hepatic ROS was decreased significantly (P<0.05). (3) As for the meat quality,48 h drip loss and shear force of breast and leg muscle were significantly decreased in broilers of 30-50 mg/kg dietary D-TOS supplementation groups, and also the cooking loss of leg muscle. But no significant differences were observed on pH45 min, pH24 h,24 h drip loss and meat color (P>0.05).Secondly, we probed into the mitigative effect of D-TOS in broilers under acute immunological stress challenged by LPS in antioxidant and non-antioxidant perspective, moreover, with the expectation to further explore the correlative mechanisms of D-TOS in depressing the production of inflammatory mediators depending on protein kinase C (PKC) and NF-κB pathway at protein levels. The effect of DL-TOA and D-TOS was compared with 288 broiler chicks of day old. A 2x4 factorial arrangement of treatments was used in which VE (30mg/kg DL-TOA, or 10,30,50 mg/kg D-a-TOS) and immunological challenge were the factors. Half birds from each treatment were challenged with 0.9% NaCl solution or LPS (250 μg/kg body weight) intraperitoneally in the lower abdominal region at 16,18 and 20 d of age. All the birds were slaughtered in 2-4 hours post-injection of the last one. Assay of splenic T and B (LTR), plasma and hepatic antioxidant indexes (a-TOH, GSH and MDA concentrations) and hepatic ROS levels were the same as mentioned above. Plasma cortisol (Cort) and prostaglandin E2 (PGE2) were analyzed by radioimmunoassay, as well as inflammatory cytokines in serum and spleen tissue, such as interferon (IFN)-y, interleukin (IL)-1(3, IL-2, IL-6 and IL-4, IL-10 concentrations. Expression and location of hepatic and splenic NF-κB were measured by fluorescent immunohistochemistry. Hepatic and splenic p65 protein in the nuclear fraction and I-κBα protein in the cytoplasm were detected by Western blotting, as well as PKC activity by ELISA assay. Moreover, hepatic and splenic histopathological changes of the liver were evaluated. The results indicated that,(1) Concerning the cellular immune response, antioxidant function, and histopathology, D-TOS (30 or 50 mg/kg) effectively enhanced the LPS-induced elevated splenic LTR and counteracted LPS-induced plasma and hepatic MDA level and antioxidant enzyme activities depletion as GSH-Px and GSH contents. Pretreatment with 50 mg/kg D-TOS markedly increased the concentrations of serum or hepatic a-TOH and also reversed LPS-induced plasma and hepatic Cort and PGE2 elevation as well as T-SOD activities. Meanwhile, degree of immunological hepatic and splenic injury was attenuated to some extent according to the histomorphology observation.(2) In the aspect of cytokins and its relative mechanisms, compared with LPS unchallenged, injection of LPS increased the levels of plasma and splenic cytokins, hepatic ROS, hepatic or splenic PKC activities, significantly, regardless of D-TOS treatment. When LPS-challenged birds pretreated with 50 mg/kg D-TOS, the increases of plasma and splenic concentrations of IFN-y, IL-1(3, IL-2, IL-6, IL-4, and IL-10 were dramatically attenuated (P<0.05 or P<0.01), and the decrease in plasma IL-2, IL-4, IL-6 and splenic IL-2, IL-6, IL-10 concentrations was also observed in the 30 mg/kg D-TOS group (P<0.05 or P<0.01). Meanwhile, significant decrease of hepatic ROS and hepatic or splenic PKC activities was found in birds pretreated with 30 or 50 mg/kg D-TOS. Furthermore, D-TOS inhibited the activation of NF-κB by preventing the degradation of IκBα.Thirdly, an attempt was made to explore the protective effects of D-TOS on the intestinal mucosa apoptosis induced by dietary oxidized rancid oil through mitochondrium pathway in broilers and its correlative mechanisms in mucosal immune response. Meanwhile, the comparision with DL-TOA and D-TOA was evaluated. A 2×5 factorial design was adopted. Two isonitrogenous experimental diets containing fresh or oxidized fish oil with different a-TOH types (30 mg/kg DL-TOA, group DL-TOA; 30 mg/kg D-TOA, group D-TOA; 15,30,60 mg/kg D-TOS, group D-TOS 1, D-TOS2 and D-TOS3) were fed to birds. The oxidized lipid used had a peroxide value of 24.9 mEq.kg-1 oil. On the termination of 42 day of age, plasma a-TOH and lipid peroxidation (LPO) concentrations were determined, and the balance of cellular oxido-reduction steady status, activities of enzymes relative to oxidative damage were determined. Moreover, mucosa of jejunum and duodenum was taken, and the mitochondria were isolated by differential speed centrifugation. For the detection of apoptosis in situ, terminal deoxynucleotidyl transferase-mediated nick end labeling staining (TUNEL) was used. The ultra-structure of mucosa epithelium was observed by Transmission Electron Microscope (TEM). Mitochondria Na+/K+-ATPase activities, cytochrome C (Cyt C) and a-TOH content, as well as activities of caspase-3, myeloperoxidase (MPO), soluble intercellular adhesion molecule (sICAM-1), IL-10, secretory immunoglobulin A (sIgA) contents in homogenate were measured in each intestinal tissue section. The results showed that,(1) Compared with the fresh oil groups, diets with the oxidized rancid oil increased the feed gain ratio (F/G) (P=0.001) of broilers at 21 day of age. Concentrations of plasma a-TOH (P=0.001), GSH of jejunum and duodenum tissues (P=0.001; P=0.001), also activities of glucose-6-phosphate dehydrogenase (G-6-PD) (P=0.0001; P=0.0001), GSH-Px (P=0.598; P=0.009) and SOD (P=0.002; P=0.029) were decreased significantly, and the activities of catalase (CAT) (P=0.001,P=0.0001) and the concentrations of LPO (P=0.004; P=0.03) were elevated; Activity and fluidity of mitochondria membrane, and Na+/K+-ATPase activity, which induced the release of Cyt C from mitochondria, and the increase of tissue caspase-3 activities (P=0.001), combined with TUNEL, showed an increase on apoptosis in mucosa epithelium (P=0.001). Moreover, dietary oxidized rancid oil increased contents of mucosal sICAM-1 (P= 0.022; P= 0.001), IL-10 and MPO activity, decreased slgA contents was obsvered (P= 0.001; P= 0.001).(2) Among the different types of 30 mg/kg a-TOH groups in the oxidized oil treatment, there was a significant decrease in duodenal LPO concentration, apoptosis ratio, and duodenum caspase-3 activity (P<0.05 or P<0.01) of group D-TOA or D-TOS in comparison with group DL-TOA, while plasma and mitochondrion a-TOH, duodenal GSH, and fluidity of mitochondria membrane showed a significant increase (P<0.05 or P<0.01), also, significant improvement of jejunum and duodenum sIgA was observed in group D-TOA and D-TOS (P<0.05).(3) Among the D-TOS groups treated with oxidized rancid oil,30 or 60 mg/kg groups significantly improved the plasma a-TOH, mitochondria a-TOH and Cyt C contents, jejunum Na+/K+-ATPase activity, jejunum and duodenum GSH, sIgA concentrations, GSH-Px and SOD activity compared with 15 mg/kg group, also, there was a significant decrease in jejunum and duodenum LPO levels, IL-10, and CAT, MPO activities, apoptosis ratio, and duodenum caspase-3 activity (P<0.05 or P<0.01). Also, there was significant difference between 30 and 60 mg/kg group in a-TOH content, apoptosis ratio, and sIgA in jejunum and duodenum (P<0.05). (4) TEM results showed that,60 mg/kg D-TOS treatment could alleviate the injury and continued apoptosis induced by oxidized rancid oil, which appeared as arrangement of villi of intestinal mucosa were sparse, which are shorter and thinner, mitochondria were swelled, cracked and miochondria cristae were broken with indiscriminate structure.As stated above, the conclusions are as follows:1.30-50 mg/kg dietary D-TOS supplementation exerted a positive effects on the growth improvement and immunity enhancement in broiler chickens under normal conditions, and could furthure enhance the antioxidant capacity of broilers, and further the water holding capacity and tenderness, which might result from increased retention of serum and hepatic a-tocopherol and reduction in lipid peroxidation, as evidenced by the decrease in MDA and ROS.2. In conclusion, the alleviated effects of 30 or 50 mg/kg D-TOS on immunological stress was not by depressing the immunity system, but by improving a-TOH availability, balancing the oxidation-reduction dynamic state. The beneficial role may depend on suppressing the production of various plasma and splenic inflammatory mediators through inhibiting of NF-κB activation and by blocking ROS signaling, in which PKC may play an assistant role.3. In conclusion, a significant oxidative stress and disturbance of the balance of oxido-reduction steady status induced by rancid oil occurred, increased apoptosis and mucosal immunological stress was showed; 30 or 60 mg/kg D-TOS supplementation may play an important role in protecting the intestinal mucosa damage, further the depressing of excessive apoptosis, and regulating the mucosal immunity reponse, which might result from increased absorption and utilization rate of miochondria a-tocopherol, improved miochondria structure and functions, and depressed inflammatory cell infiltration.