Effects Of Weanling Age On Oxidative-Stress In Weaned Piglets

Early weaning of piglets is a major challenge in swine production, which always causes the degradation of growth performance, reduction in feed efficiency, as well as the invasion of the exogenous pathogenic substances. Recent studies showed that serious oxidative stress was observed in weaned piglets, and induced intestinal barrier dysfunction, which is one of the most important reasons for the weaned stress syndrome. Currently,21d of weaning age was widely used in intensive swine production, and more attention has been paid to the super early weaning age of 14d.However, there are no systemic studies about the development of intestinal bacterial function changes in different weaning age piglets. In this study, the super early weaned piglets (14d of age) and the early weaned piglets (21d of age) were used as weaned stress model, to compare the changes of antioxidant abilities, immunity and intestinal flora between 14d and 21d age of weaned piglets. The mechanism of intestinal barrier function developmentwas investigated to study the apoptosis in super early weaned piglets. The results will provide the theoretical basis for relieving piglet weaning syndromeaccording to the intestinal barrier developmentcharacteristics in different weaning age of weaned piglets.1. Influence of different weaning age on antioxidant capacity in pigletsFifty new born piglets from 5 litters (10 piglets per litter) were selected in this study. At 14d and 21 d old of age, two piglets from each litter were selected and divided into 14d weaning group and 2Id weaning group by litter (2 piglets/pen). The other piglets were remaining suckling. At 18d and 25d old of age, one piglet per litter from 14d and 21d weaning group were selected and slaughtered for further study. And one piglet from the remaining suckling piglets was also slaughtered at the same age. Activities of SOD and GSH-PX, contents of MDA, NO, and H2O2 in serum and different parts of intestinal tissues were measured. The contents of DAO, IFABP and cortisol in serum were also measured. Results showed that, concentrations of cortisol were significantly increased in 14-d and 21d old weaned piglets compared to their corresponding suckling groups (P< 0.05), which indicated that weaning induced stress in weaned piglets. While the concentrations of cortisol in 21d weaned piglets were higher than that in 14d weaned piglets, whichshowed that the higher weaning age, the more sensitivity to weaning stress. Antioxidant capacity was decreased in 14d and 21d weaned piglets, however, no significant differences of GSH-Px activity and MDA, NO, and H2O2 contents were observed between 14d weaned piglets and 14d old suckling piglets. While the antioxidant capacity of 21d weaned piglets was higher than that in the 14d weaned piglets, which indicated that antioxidant capacity increases with the increase of age. No differences in DAO and IFABP contents were observed in 14d and 21d old weaned pigs (P> 0.05).Both of the 14d and 21d weaning can decrease the piglets’antioxidant ability in serum, while no significant differences (P> 0.05) in GSH-Px activity and MDA, H2O2, and NO contents were observed in serum of 14d weaned pigs. The serum antioxidant ability in 21 d weaned pigs were higher (P< 0.05) than that in 14d weaned pigs, which indicated that antioxidant capacity increases with the age, and the antioxidant capacity developed relatively fully in 21 d weaned pigs. Contents of MDA, H2O2, and NO in ileum of 14d weaned piglets were significantly higher compared to 14d old suckling pigs (P< 0.05), while the activities of SOD and GSH-Px decreased in rectum and cecum, and the H2O2and NO contents were increased in rectum (P< 0.05). All these indicated that super early weaning can also decrease the antioxidant capacity in serum and gut, the antioxidant capacity was obviously decreased in gut, which might indicated that super early weaning had a direct impact on gut. In addition, the contents of H2O2and NO in 21d weaned pigs were lower than that in the 14d weaned pigs, which might infer that the 14-d weaned pigs are more susceptible to free radical.2. Effect of different weaning age on immunity response in pigletsBased on the results of the first part of this study, the present study was to investigate the effect of different weaning age on immunity response in piglets. In this part, the immune organ index of piglets were measured, contents of IL-2, IL-4, IL-6, IgM, and IL-1(3 in serum were determined by ELISA. Quantitative real time PCR was used to analyze the gene expressions of IL-2, IL-4, IL-6, and IL-iβ in spleen. Results showed that, weaning age had no significant differences on immune organ index, IgM and IL-4concentrations (P> 0.05). Serum IL-2 contents were significantly higher (P< 0.05) in 14d weaned pigs than that in the 14d suckling group, but it is lower than that in 21d weaned pigs(P< 0.05). No significant difference (P> 0.05)in IL-6 concentrations in serum were observed between 14d weaned pigs and 14d suckling pigs, the content of IL-6 in 21d old weaned pigs were higher than that in 21d old suckling pigs (P< 0.01), and it was also significantly higher than that in 14d old weaned pigs(P< 0.01). Quantitative real time PCR results showed that, gene expression of IL-6, IL-4, and IL-2 increased in 14d old weaned pigs (P< 0.05), while no significant changes of IL-1β expression in 14d old weaned pigs and IL-2 expression in 21d old weaned pigs were observed (P> 0.05). Results indicated that serum cytokine levels changed in different degrees, compared to the pigs weaned in 21d old, pigs weaned in 14d old showed better immunity response.3. Effect of different weaning age on gut bacterial quantity in weaned pigsThe third study was to investigate the effect of different weaning age on gut bacterial population in weaned pigs based on the two studies above. Quantitative real time PCR was used to analyze the main bacterial amount in the four treatments. Results showed that, the amount of lactobacillus decreased in ileum, rectum, and cecum in 14d and 21d old weaned pigs compared with the control (P< 0.05). The amount of bifidobacterium also decreased in cecum in 14d and 21d old weaned pigs (P< 0.05). The amount of lactobacillus and bifidobacterium increased in different parts of gut, and had a significant positive correlation with the activity of SOD and GSH-PX, and negative correlation with the content of MDA, H2O2, and NO (P< 0.05). Results indicated that the gut barrier function was destroyed in 14d and 21d old weaned pigs, after weaning, the amount of beneficial bacterial lactobacillus and bifidobacterium decreased, while the harmful bacterial escherichia coli increased, the bacterial counts changes have significantcorrelation with gut antioxidant status and free radical levels.4. Impact and mechanism of super early weaning on piglets’intestinal apoptosisBased on the 3 studies above, in present study, the impact of super early weaning on piglets’intestinal apoptosis was investigated. HE staning, terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling (TUNEL), transmission electron microscopy (TEM), and qRT-PCR methods were conducted to characterize apoptosis and intestinal mucosal barrier damage induced by weaning in pigs. The findings showed that no significant (P> 0.05) intestinal wallthickness, villus height, and villus width in the jejunum were observed between the 14d weaned pigs and the suckling pigs. The 14d weaned pigs showed deeper crypt depths (P< 0.05) than suckling pigs. Weaning enhanced enterocytes apoptosis. In addition, TUNEL of the jejunum revealedan increased in the number of apoptotic cells in weaned pigs, with significant localization of apoptotic cells in the upper tip of the intestinal villi and lamina propria. The microvilli were widely spaced, some enterocytes showed mitochondrial ridge damage, vacuolated cytoplasm, cell compaction, condensed chromatin, and fragmented mitochondria. While in the control suckling pigs, the macrovilli arranged closely, cells connected well, mitochondrial forms normal, several cells showed mitochondria damage, condensed chromatin. Weaning also induced up-regulation of Bax, caspase-3, caspase-8, caspase-9, and Fas (P<0.05), and down-regulation of anti-apoptosis gene (Bcl2, P<0.05). Quantitative reverse transcription-PCR analyses indicate that weaning induced the apoptosis associated Fas-signaling and mitochondrial pathways in weaned pigs.Some conclusions were drawn as follows:(1) Both the weaning age of 14d and the 21d can decrease the antioxidant capacity, increase lipid peroxidation and the contents of H2O2 and NO in piglets’ gut, and the antioxidant ability could increase with the increase of age. The antioxidant capacity developed relatively fully in 21 d weaned pigs. The 14d weaned pigs are more susceptible to free radical.(2) Due to the different weaned period, serum cytokine levels changes in different degrees, weaning can activate the active immunity, but it also decrease the immune response, increase the release of inflammatory factors. Compared to the pigs weaned in 21d old, pigs weaned in 14d old shows better immunity response.(3) The redox of intestine changes in different weaning age, and induced the dysfunction of gut bacteria. After weaning, the amount of harmful bacterial escherichia coli increased while the beneficial bacterial lactobacillus and bifidobacterium decreased. The bacterial amount changes have significant correlation with gut antioxidant status and free radical levels.(4) The super early weaning can enhance the enterocytes apoptosis, destroy the enterocytes integrity. Weaning induced the apoptosis associated Fas-signaling and mitochondrial pathways in weaning pigs.