Effect of 7-Nitroindazole Sodium on the Cellular Distribution of Neuronal Nitric Oxide Synthase in the Cerebral Cortex of Hypoxic Newborn Piglets

Cerebral hypoxia results in generation of nitric oxide (NO) free radicals by Ca⁺⁺-dependent activation of neuronal nitric oxide synthase (nNOS). The present study tests the hypothesis that the hypoxia-induced increased expression of nNOS in cortical neurons is mediated by NO. To test this hypothesis the cellular distribution of nNOS was determined immunohistochemically in the cerebral cortex of hypoxic newborn piglets with and without prior exposure to the selective nNOS inhibitor 7-nitroindazole sodium (7-NINA). Studies were conducted in newborn piglets, divided into normoxic (n = 6), normoxic treated with 7-NINA (n = 6), hypoxic (n = 6) and hypoxic pretreated with 7-NINA (n = 6). Hypoxia was induced by lowering the FiO₂ to 0.05–0.07 for 1 h. Cerebral tissue hypoxia was documented by decrease of ATP and phosphocreatine levels in both the hypoxic and 7-NINA pretreated hypoxic groups (P < 0.01). An increase in the number of nNOS immunoreactive neurons was observed in the frontal and parietal cortex of the hypoxic as compared to the normoxic groups (P < 0.05) which was attenuated by pretreatment with 7-NINA (P < 0.05 versus hypoxic). 7-NINA affected neither the cerebral energy metabolism nor the cellular distribution of nNOS in the cerebral cortex of normoxic animals. We conclude that nNOS expression in cortical neurons of hypoxic newborn piglets is NO-mediated. We speculate that nNOS inhibition by 7-NINA will protect against hypoxia-induced NO-mediated neuronal death.     

Does the environmental background (intensive v. outdoor systems) influence the behaviour of piglets at weaning?

Under intensive pig husbandry, outdoor systems offer a more complex physical and social environment compared with indoor systems (farrowing sheds). As the rearing environment affects behavioural development, it can, therefore, influence behavioural responses of pigs to stressful environments in later stages of production. We tested how the rearing environment influenced behavioural responses to a novel arena test in piglets on the day that they were weaned and mixed into large groups. We recorded video footage and compared the behavioural responses of 30 outdoor-raised and 30 farrowing shed-raised piglets tested in an experimental arena and sequentially exposed to four challenges (each for 5 min) on the day of weaning. Quantitative and qualitative behavioural measures were recorded using time budgets and scoring demeanour or ‘qualitative behavioural expression’ (using Qualitative Behavioural Assessment (QBA)). When held in isolation (challenge 1), both groups were scored as more ‘scared/worried’, while outdoor-raised piglets spent more time eating and jumping against the arena walls. Both groups interacted with a plastic ball (challenge 2: exposure to a novel object) during which they were scored as more ‘playful/curious’ than other challenges. When a food bowl was introduced (challenge 3), farrowing shed-raised piglets were more interested in playing with the food bowl itself, whereas outdoor-raised piglets spent more time eating the feed. Finally, there were no significant differences in social behaviour (challenge 4: introduction of another piglet) between the two groups in terms of the latency to contact each other, amount of time recorded engaged in aggressive/non-aggressive social interactions or QBA scores. Although piglets spent 30% of their time interacting with the other piglet, and half of this time (47%) was engaged in negative interactions (pushing, biting), the levels of aggression were not different between the two groups. Overall, outdoor-raised piglets ate more and were scored as more ‘calm/passive’, whereas farrowing shed-raised piglets spent more time investigating their environment and were scored as more ‘playful/inquisitive’. In conclusion, we did not find differences in behaviour between outdoor-raised and farrowing shed-raised piglets that would highlight welfare issues. The differences found in this study may reflect conflicting affective states, with responses to confinement, neophobia and motivation for exploration evident.     

Kinetics of changes in gene and microRNA expression related with muscle inflammation and protein degradation following LPS-challenge in weaned piglets

To test the dynamic changes of the expression of genes and microRNA in the gastrocnemius muscle after LPS challenge, 36 piglets were assigned to a control group (slaughtered 0 h after saline injection) and LPS groups (slaughtered at 1 h, 2 h, 4 h, 8 h, and 12 h after LPS treatment, respectively). After LPS treatment, the mRNA expression of IL-1β, IL-6, and TNF-α reached maximal levels at 1 h, 2 h, and 1 h, respectively (P < 0.05), and mRNA expression of TLR4, NODs, muscle-specific ring finger 1, and muscle atrophy F-box peaked at 12 h (P < 0.05). Moreover, the expression of miR-122, miR-135a, and miR-370 reduced at 1 h, 1 h, and 2 h, respectively (P < 0.05), and miR-34a, miR-224, miR-132, and miR-145 reached maximum expression levels at 1 h, 1 h, 2 h, and 4 h, respectively (P < 0.05). These results suggested that mRNA expression of pro-inflammatory cytokines was elevated in the early stage, mRNA expression of genes related to TLR4 and NODs signaling pathways and protein degradation increased in the later phase, and the expression of microRNA related to muscle inflammation and protein degradation changed in the early stage after LPS injection.     

Validation of Brix refractometer to estimate colostrum immunoglobulin G content and composition in the sow

Colostrum is an essential source of immunoglobulin G (IgG) for neonate piglets. However, colostrum IgG content and nutritional composition can vary considerably among sows due to age, parity, feeding regime and immunological background. Currently, there is no practical way to obtain information about colostrum IgG concentration at herd level. We evaluated sows’ colostrum IgG content on-farm using a Brix refractometer and its performance was compared with that of an IgG ELISA. In addition, nutritional compositions of the colostrum samples were analyzed using Fourier transform IR spectroscopy. Colostrum samples (5 to 6 ml) (n=153) were obtained within 0 to 3 h of farrowing. However, to obtain a 24 h IgG profile for 11 sows, colostrum samples were collected at 0, 2, 4, 6, 8, 10, 16 and 24 h after farrowing. A 0.3 ml of freshly drawn colostrum sample was used for the on-farm measurement of Brix percentages using a digital refractometer shortly after collection. The remaining fractions of the samples were frozen and submitted to laboratory analysis for total IgG, using a commercially available pig IgG ELISA kit. For nutritional composition analysis, a 35 ml colostrum sample (n=34) was obtained immediately after birth of first piglet from the first three pairs of frontal teats. Colostrum concentrations of IgG averaged 52.03±30.70 mg/ml (mean±SEM) at 0 to 3 h after farrowing. Concentration of IgG decreased on average by 50% during the 1st day of lactation (P<0.01). Sow parity did not influence colostrum concentrations of IgG. Differences in colostrum composition were recorded between two herds and among the parity groups (P<0.05). The Brix refractometer measurement of colostrum and the corresponding log transformed IgG measurements from the ELISA were moderately correlated (r=0.63, P<0.001, n=153). Based on the classification we suggest here, low levels of IgG (14.5±1.8 mg/ml) were recorded for colostrum samples with Brix readings below 20%. Borderline colostrum IgG content (43.8±2.3 mg/ml) had Brix readings of 20% to 24%, adequate colostrum IgG content (50.7±2.1 mg/ml) had Brix % readings of 25% to 29% and very good IgG colostrum content (78.6±8.4 mg/ml) had Brix readings >30%. Colostrum IgG concentration is highly variable among sows, Brix measurement of a sows’ fresh colostrum is an inexpensive, rapid and satisfactorily accurate method of estimating IgG concentration, providing indication of differentiation between good and poor IgG content of colostrum.     

表达ST1-LTB-a-b融合蛋白基因工程菌株的构建及其免疫原性分析

采用分子生物学技术构建了含有ST1-LTB-a-b融合基因的重组菌株BL21(DE3)(pETST3LTBab), SDS-PAGE和 Western blotting分析表明ST1-LTB-a-b融合基因在大肠杆菌中得到了高效表达, 融合蛋白分子量约为110 kD; 20 L发酵罐培养得到的最佳诱导条件为: 重组菌株以1%接种量、5 L/min通气量培养3 h后加终浓度为0.03 mol/L的乳糖诱导, 通气量升至12.5 L/min继续培养6 h, 表达量占菌体总蛋白的38.53%; 表达的ST1-LTB-a-b融合蛋白无毒性但具有免疫原性, 可以抵抗大肠杆菌和产气荚膜梭菌的感染; 构建的重组菌株BL21(DE3)(pETST3LTBab)有望作为预防仔猪腹泻基因工程疫苗的候选生产菌株。     

Presence of 22-kDa protein reacting with sera in piglets experimentally infected with Brachyspira hyodysenteriae

In order to examine the effect of spectinomycin on outbreaks of swine dysentery, experimental infection of piglets with Brachyspira hyodysenteriae was carried out. Feed with and without spectinomycin (SP) was given to each piglet ad libitum and the susceptibility of the piglets to infection with B. hyodysenteriae was compared between SP-treated and untreated piglets. The results showed that the SP-treated piglets did not display clinical signs of swine dysentery unlike the untreated piglets. The sera obtained from these piglets were examined by the microscopic agglutination test and antibodies to B. hyodysenteriae in both groups of experimentally infected piglets were detected and the reaction was serogroup-specific. The agglutination titers were very high in the untreated piglets with dysentery while the titers in the SP-treated piglets were lower than those in the untreated piglets. In addition, the immunoblotting technique was applied and the results demonstrated that 22- and 17-kDa proteins in strain ATCC 31212 (serogroup B) reacted strongly with the sera from the untreated piglets but not with the sera from the SP-treated piglets. The 22- and 17-kDa proteins also reacted with strain ATCC 27164 (serogroup A) which belongs to a different serogroup. The 22- and 17-kDa proteins were also confirmed in six other strains of B. hyodysenteriae which belong to six different serogroups. These proteins were sensitive to proteinase K. These results indicate that the 22- and 17-kDa proteins are common to eight strains of B. hyodysenteriae which differ serologically from each other.