Reducing agent can be omitted in the incubation medium of the batch in vitro fermentation model of the pig intestines.

Over the past decade, in vitro methods have been developed to study intestinal fermentation in pigs and its influence on the digestive physiology and health. In these methods, ingredients are fermented by a bacterial inoculum diluted in a mineral buffer solution. Generally, a reducing agent such as Na2S or cysteine-HCl generates the required anaerobic environment by releasing metabolites similar to those produced when protein is fermented, possibly inducing a dysbiosis. An experiment was conducted to study the impact of two reducing agents on results yielded by such in vitro fermentation models. Protein (soybean proteins, casein) and carbohydrate (potato starch, cellulose) ingredients were fermented in vitro by bacteria isolated from fresh feces obtained from three sows in three carbonate-based incubation media differing in reducing agent: (i) Na2S, (ii) cysteine-HCl and (iii) control with a mere saturation with CO2 and devoid of reducing agent. The gas production during fermentation was recorded over 72 h. Short-chain fatty acids (SCFA) production after 24 and 72 h and microbial composition of the fermentation broth after 24 h were compared between ingredients and between reducing agents. The fermentation residues after 24 h were also evaluated in terms of cytotoxicity using Caco-2 cell monolayers. Results showed that the effect of the ingredient induced higher differences than the reducing agent. Among the latter, cysteine-HCl induced the strongest differences compared with the control, whereas Na2S was similar to the control for most parameters. For all ingredients, final gas produced per g of substrate was similar (P>0.10) for the three reducing agents whereas the maximum rate of gas production (R max) was reduced (P0.10) after 24 h of fermentation with Na2S and in the control without reducing agent. Molar ratios of branched chain-fatty acids were higher (P<0.05) for protein (36.5% and 9.7% for casein and soybean proteins, respectively) than for carbohydrate (<4%) ingredients. Only fermentation residues of casein showed a possible cytotoxic effect regardless of the reducing agent (P<0.05). Concerning the microbial composition of the fermentation broth, most significant differences in phyla and in genera ascribable to the reducing agent were found with potato starch and casein. In conclusion, saturating the incubation media with CO2 seems sufficient to generate a suitable anaerobic environment for intestinal microbes and the use of a reducing agent can be omitted.

Adding mucins to an in vitro batch fermentation model of the large intestine induces changes in microbial population isolated from porcine feces depending on the substrate.

Adding mucus to in vitro fermentation models of the large intestine shows that some genera, namely lactobacilli, are dependent on host-microbiota interactions and that they rely on mucosal layers to increase their activity. This study investigated whether this dependence on mucus is substrate dependent and to what extent other genera are impacted by the presence of mucus. Inulin and cellulose were fermented in vitro by a fecal inoculum from pig in the presence or not of mucin beads in order to compare fermentation patterns and bacterial communities. Mucins increased final gas production with inulin and shifted short-chain fatty acid molar ratios (P < 0.001). Quantitative real-time PCR analyses revealed that Lactobacillus spp. and Bifidobacterium spp. decreased with mucins, but Bacteroides spp. increased when inulin was fermented. A more in-depth community analysis indicated that the mucins increased Proteobacteria (0.55 vs 0.25%, P = 0.013), Verrucomicrobia (5.25 vs 0.03%, P = 0.032), Ruminococcaceae, Bacteroidaceae and Akkermansia spp. Proteobacteria (5.67 vs 0.55%, P < 0.001) and Lachnospiraceae (33 vs 10.4%) were promoted in the mucus compared with the broth, while Ruminococcaceae decreased. The introduction of mucins affected many microbial genera and fermentation patterns, but from PCA results, the impact of mucus was independent of the fermentation substrate.

Forage plants as an alternative feed resource for sustainable pig production in the tropics: a review.

Globally, pressure on concentrate feed resources is increasing, especially in the tropics where many countries are net importers of food. Forage plants are a possible alternative, but their use as feed ingredients for pigs raises several issues related to their higher fibre and plant secondary metabolites contents as well as their lower nutritive value. In this paper, the nutritive value of several forage species and the parameters that influence this nutritive value in relationship to the plant family, the physiological stage, the plant part and the preservation method (fresh, hay and silage) are reviewed. The influence of the breed and the physiological status of the animal on animal voluntary intake of fibre-rich ingredients, digestibility as related to gastrointestinal volume and transit time and growth performances are also discussed. The final section highlights the advantages and drawbacks of forage plants in pig diets and stresses the need for proper economic evaluation to conclude on the benefits of the use of forage plants in pig feed.

Use of medium without reducing agent for in vitro fermentation studies by bacteria isolated from pig intestine.

Over the past decade, several in vitro methods have been developed to study intestinal fermentation in pigs and its influence on health. In these methods, samples are fermented by a bacterial inoculum diluted in a mineral buffer solution. Generally, a reducing agent such as Na(2)S or cysteine HCl generates the required anaerobic environment by release of H(2)S inducing an imbalance among bacterial species by the production of toxic metabolites. Therefore, an experiment was conducted to study the impact of reducing agent on fermentation patterns. Protein (soybean protein and/or casein) and carbohydrate (potato starch and/or cellulose) ingredients were fermented in vitro by pig intestinal bacteria from fresh feces obtained from 3 sows fed an antibiotic-free commercial diet in 3 incubation media differing in reducing agent: (i) Na(2)S, (ii) cysteine HCl, or (iii) without reducing agent. Gas fermentation kinetics were monitored over 72 h (pressure was measured every 2 min). Short-chain fatty acid (SCFA) production after 24 and 72 h were compared among ingredient and reducing agents (n = 2). Gas production was higher (P < 0.05) when fermenting carbohydrate than protein ingredients. Except for soybean protein, total SCFA production after 24 and 72 h was similar (P > 0.05) for each ingredient regardless the incubation medium. The SCFA molar ratios did not differ (P > 0.05) between Na(2)S and without reducing agent. In conclusion, saturation of incubation media with CO(2) seems sufficient to generate an anaerobic environment. So incubation media could be simplified by omitting the reducing agent without influencing the fermentation kinetics and SCFA production.

Discrepancies in microbiota composition along the pig gastrointestinal tract between in vivo observations and an in vitro batch fermentation model.

In vitro fermentation models are increasingly used to assess prebiotic potential of novel indigestible carbohydrates (CHO). A trial was performed to assess the validity of such approaches by comparing the influence of fermentation of inulin and cellulose on microbiota in vivo and in vitro. Two semipurified diets based on 5% inulin or 5% cellulose were fed to 2 groups of four 25-kg pigs. After 3 wk, the pigs were slaughtered and digesta was sampled from jejunum, ileum, cecum, and 3 parts of the colon to measure pH and microbiota population. An in vitro gas fermentation test was also performed on inulin and cellulose using fresh feces of the experimental pigs as bacterial inoculum. The gas production kinetics were modeled and fermentation broth sampled after 5, 8, 12, 24, and 72 h. Bacterial DNA was extracted and quantitative PCR was performed to quantify total bacteria, lactobacilli, bifidobacteria, Bacteroides, Clostridium cluster I, and Escherichia coli. Total bacteria quantification was similar between both systems. In vivo, total bacteria increased (P < 0.001) along the gut until the second part of the colon (from 10.5(7) to 10(10) cfu/mg) and then decreased (P < 0.05) to 10(9) cfu whereas in vitro, it increased (P < 0.05) until 12 to 24 h of fermentation (from 10(9) to 10.5(9) cfu/mL) and then decreased (P < 0.05) to initial level (10(9) cfu/mL). This evolution was consistent with fermentation kinetics. In both models, inulin increased (P < 0.05) the ratio of bifidobacteria and E. coli populations in the total microflora compared to cellulose. However, in vivo this was observed only in the first parts of the gut whereas in vitro the effect lasted for 72 h. Inulin also increased (P < 0.001) Bacteroides genus in vitro but not in vivo where the evolutions of Bacteroides were similar (P > 0.05) for both CHO. Evolutions of lactobacilli and Clostridium populations in both systems were also not consistent. This can be ascribed to specific bacterial properties as, for example, adhesive properties or sensitivity to sulfur reducing agent used in the in vitro model. As is, the in vitro model does not reflect properly changes in microbiota along the digestive tract induced by specific feed ingredients compared to in vivo observations.

Nutritional properties of potato protein concentrate compared with soybean meal as the main protein source in feed for the double-muscled Belgian Blue bull.

The objective of this experiment was to compare the nutritional properties of potato protein concentrate, a by-product of the starch industry produced entirely in Europe, with that of soybean meal (SBM), for growing cattle. The experiment was conducted on double-muscled Belgian Blue bulls, fitted with rumen, duodenal and ileal cannulas, according to a 4 × 4 Latin square design. They were fed three different iso-N and iso-net energy diets formulated according to the Dutch feed evaluation system, differing in the nature of the main protein source, which was either SBM ('SBM' treatment), potato protein concentrate (PPC, 'PPC' treatment) or an iso-N mixture of these two protein sources ('mixed' treatment). A fourth treatment consisted of 'PPC' supplemented by 9.5% digestible proteins supplied by duodenal perfusion of sodium caseinate (CAS, 'PPC + CAS' treatment). No significant difference was observed in the ruminal fluid pH, whereas both 'PPC' and 'PPC + CAS' had the effect of reducing the ruminal ammonia nitrogen (N-NH3) concentration. No significant difference was observed in the apparent intestinal digestibility of the dry matter (DM), organic matter (OM) or N. Outflows of non-NH3-N, microbial proteins and dietary proteins from the rumen were similar for 'PPC', 'SBM' and 'mixed', and increased with CAS infusion by 20%, 17% and 27%, respectively. On the basis of in vivo observations, the degradability of SBM and PPC proteins was estimated at 0.60 and 0.43, respectively, corresponding to the values quoted in the literature. The supply of digestible essential amino acids (EAA) was significantly greater with 'PPC + CAS' and did not differ among 'SBM', 'mixed' and 'PPC'. This illustrates the difficulty of altering the amino acid (AA) pattern of digestible protein by the nature of the protein of dietary origin when an animal is fed a high nutritional value diet. N retention was not affected by replacing SBM with PPC, but increased by 10% with CAS infusion. On the basis of the plasma AA pattern, the supply of digestible Met was probably limiting with 'SBM', 'mixed' and 'PPC'. The CAS perfusion supplemented all AA, including Met, leading to increased N retention. This improvement was limited, however, and N utilisation remained unchanged between treatments. In conclusion, despite a more favourable EAA pattern, PPC offered no advantage compared with SBM for growing bulls when diets were formulated according to the Dutch feed evaluation system.

Effect of a bovine colostrum whey supplementation on growth performance, faecal Escherichia coli population and systemic immune response of piglets at weaning.

This study examined the effect of a bovine colostrum whey supplementation on growth performance, feed intake, faecal Escherichia coli population and systemic immune response of piglets at weaning. A total of 96 piglets weaned at 26 ± 2 days of age were assigned for 4 weeks to one of the two treatments: (1) the control (commercial diet with bovine milk whey powder) and (2) the colostrum (commercial diet with freeze-dried bovine colostrum whey) treatments. The two supplements were incorporated in the diet at a level of 20 g/kg during the first 2 weeks after weaning and lowered to a level of 10 g/kg for the next 2 weeks. BW and feed intake were measured weekly. Faecal E. coli counts were determined weekly on specific culture media. Blood samples were collected weekly and submitted to a cell counter analyser for their main components (red and white blood cells, platelets) and flow cytometry was used to determine the lymphocyte population (B, T, Th and Tc). Finally, total seric immunoglobulin (IgM, IgG and IgA) concentrations were determined by the ELISA method. During the first week of the trial, the piglets from the colostrum treatment had improved average daily gain (170 g/day v. 81 g/day, P < 0.001), average daily feed intake (346 g/day v. 256 g/day, P = 0.03) and feed efficiency (BW gain/feed intake) (0.48 v. 0.31, P = 0.04). The pigs fed the colostrum treatment had also a 25% increase in circulating IgA (P = 0.03) compared with the control treatment the first week. It is concluded that a distribution of bovine colostrum whey (20 g/kg diet) during the first week post-weaning induces a systemic IgA response and has a beneficial action on growth performances and feed efficiency.

The A5 gene of alcelaphine herpesvirus 1 encodes a constitutively active G-protein-coupled receptor that is non-essential for the induction of malignant catarrhal fever in rabbits.

Many gammaherpesviruses encode G-protein-coupled receptors (GPCRs). Several in vivo studies have revealed that gammaherpesvirus GPCRs are important for viral replication and for virus-induced pathogenesis. The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) is carried asymptomatically by wildebeest, but causes malignant catarrhal fever (MCF) following cross-species transmission to a variety of susceptible species. The A5 ORF of the AlHV-1 genome encodes a putative GPCR. In the present study, we investigated whether A5 encodes a functional GPCR and addressed its role in viral replication and in the pathogenesis of MCF. In silico analysis supported the hypothesis that A5 could encode a functional GPCR as its expression product contained several hallmark features of GPCRs. Expression of A5 as tagged proteins in various cell lines revealed that A5 localizes in cell membranes, including the plasma membrane. Using [35S]GTPgammaS and reporter gene assays, we found that A5 is able to constitutively couple to alpha i-type G-proteins in transfected cells, and that this interaction is able to inhibit forskolin-triggered cAMP response element-binding protein (CREB) activation. Finally, using an AlHV-1 BAC clone, we produced a strain deleted for A5 and a revertant strain. Interestingly, the strain deleted for A5 replicated comparably to the wild-type parental strain and induced MCF in rabbits that was indistinguishable from that of the parental strain. The present study is the first to investigate the role of an individual gene of AlHV-1 in MCF pathogenesis.

Effects of oral supplementation with bovine colostrum on the immune system of weaned piglets.

The aim of this study was to evaluate the influence of bovine colostrum supplementation on the immune system of weaned piglets in a context of a full ban of in-feed antibiotics. After weaning at 21 days, 24 outbred piglets were fed with a diet supplemented daily for three weeks with 0, 1 or 5 g of colostrum. Feed intake, growth performance, haematological parameters, and serum and local anti-colostrum immunoglobulin levels were examined. Lymphocytes from the blood, spleen, and gut-associated lymphoid were analysed for phenotype as well as for their ability to produce cytokines. The stimulation index (SI) of mononuclear cells from different organs was obtained after colostral or mitogenic stimulation. Feed intake, growth, and haematological parameters were not significantly affected by colostrum. Total serum IgA levels were increased after colostrum supplementation, with a transient decrease in total IgG. Local anti-colostrum immunization was observed in colostrum-fed piglets. The CD21+/CD3+ cells populations of the ileal Peyer's patch (iPP) were markedly affected. The SI of lymphocyte populations changed significantly whereas, naive blood lymphocytes were not stimulated in vitro in the presence of bovine colostrum, suggesting local anti-colostrum immunization and an absence of direct mitogenic effects of the colostrum. Both Th1 and Th2 cytokine production was present in the different organs of colostrum-fed piglets. Bovine colostrum especially stimulated iPP cells.

[Viral homologues of G-protein-coupled receptor]. / Les homologues viraux des récepteurs cellulaires couplés aux protéines G.

G-protein-coupled receptors (GPCR) are seven transmembrane proteins that convert extracellular stimuli to cell signaling.Viral genes homologous to cellular GPCR have been described in the genome of Betaherpesvirinae, Gammaherpesvirinae and Poxviridae. The goal of this review is to summarize the knowledge available on viral GPCR (vGPCR) with a special interest for their roles in the biology and the pathogenesis of the infection. This review highlights some properties of vGPCR that are not shared by their cellular homologues and stresses the diversity of their functions in the biology of the infection.

Cloning of the genome of Alcelaphine herpesvirus 1 as an infectious and pathogenic bacterial artificial chromosome.

Alcelaphine herpesvirus 1 (AlHV-1), carried asymptomatically by wildebeest, causes malignant catarrhal fever (MCF) following cross-species transmission to a variety of susceptible species of the order Artiodactyla. The study of MCF pathogenesis has been impeded by an inability to produce recombinant virus, mainly due to the fact that AlHV-1 becomes attenuated during passage in culture. In this study, these difficulties were overcome by cloning the entire AlHV-1 genome as a stable, infectious and pathogenic bacterial artificial chromosome (BAC). A modified loxP-flanked BAC cassette was inserted in one of the two large non-coding regions of the AlHV-1 genome. This insertion allowed the production of an AlHV-1 BAC clone stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. The loxP-flanked BAC cassette was excised from the genome of reconstituted virions by growing them in permissive cells stably expressing Cre recombinase. Importantly, BAC-derived AlHV-1 virions replicated comparably to the virulent (low-passage) AlHV-1 parental strain and induced MCF in rabbits that was indistinguishable from that of the virulent parental strain. The availability of the AlHV-1 BAC is an important advance for the study of MCF that will allow the identification of viral genes involved in MCF pathogenesis, as well as the production of attenuated recombinant candidate vaccines.

Automatic morphological sieving: comparison between different methods, application to DNA ploidy measurements.

The aim of the present study is to propose alternative automatic methods to time consuming interactive sorting of elements for DNA ploidy measurements. One archival brain tumour and two archival breast carcinoma were studied, corresponding to 7120 elements (3764 nuclei, 3356 debris and aggregates). Three automatic classification methods were tested to eliminate debris and aggregates from DNA ploidy measurements (mathematical morphology (MM), multiparametric analysis (MA) and neural network (NN)). Performances were evaluated by reference to interactive sorting. The results obtained for the three methods concerning the percentage of debris and aggregates automatically removed reach 63, 75 and 85% for MM, MA and NN methods, respectively, with false positive rates of 6, 21 and 25%. Information about DNA ploidy abnormalities were globally preserved after automatic elimination of debris and aggregates by MM and MA methods as opposed to NN method, showing that automatic classification methods can offer alternatives to tedious interactive elimination of debris and aggregates, for DNA ploidy measurements of archival tumours.

Influence of sample size on image cytometry of DNA ploidy measurements.

OBJECTIVE: To assess the number of nuclei required for significant image cytometry DNA ploidy measurements on one archival case of breast cancer. STUDY DESIGN: From one case of aneuploid DNA breast cancer, 18 subsets made up of 152-1,524 for the whole population of undamaged nuclei and made up of 74-735 epithelial nuclei had DNA measured. DNA ploidy type and five DNA ploidy indices, allowing DNA ploidy histogram interpretation were evaluated on each population. RESULTS: Three hundred nuclei were always sufficient for DNA typing, whereas reliable results for DNA ploidy indices required at least 750 nuclei. CONCLUSION: To DNA measure the above number of nuclei, fully automated image cytometry DNA ploidy measurements are required.

Influence of debris and aggregates on image cytometry DNA measurement of archival tumors.

OBJECTIVE: To investigate, for seven samples, the influence of unwanted elements (e.g., remains of erythrocyte cell membranes, sliced nuclei, damaged nuclei and aggregates) on image cytometry DNA ploidy measurements. STUDY DESIGN: Two normal reference tissues (brain and breast), one breast cancer and four brain tumors were studied. For each sample, the influence of the different classes of debris on DNA ploidy histograms and indices was evaluated. RESULTS: The influence differs regarding each class of debris and the index to be evaluated. CONCLUSION: Strict and precise elimination of debris and aggregates is required. Moreover, strategies and efforts that must be applied to automated elimination of these unwanted elements must be a direct function of the bias they introduce into DNA ploidy measurements.

Apoptotic death in cortical neurons of mice subjected to focal ischemia.

Permanent focal cortical ischemia was induced in mice by electrocoagulation of the middle cerebral artery. At different time intervals after the injury, the volume of infarction was assessed together with an analysis of neuronal death. Morphological studies of ischemic brains and detection of nucleosomal DNA ladder within ipsilateral cortices might implicate a component of this neuronal loss to apoptosis as well as necrosis. Furthermore, we used the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling) procedure to detect in situ DNA fragmentation. The localization and the proportion of apoptotic cells in the ischemic mouse brain would indicate that apoptosis contributes largely to the cellular loss induced by cerebral ischemia.

[Clinical and biochemical considerations on depressive states occuring during parkinsonian syndrome treated by L-Dopa]. / Considérations cliniques et biochimiques sur les états dépressifs survenant lors de syndromes parkinsoniens traités par la L Dopa

From 17 observations of depressive illness occuring to parkinsonians treated with L. Dopa alone or associated with an inhibitor of peripheral decarboxylase, several types of depressive illness can be distinguished according to their date of appearance, their evolution with or without tricyclic anti-depressant drugs tertiary amine. According to the characteristics of these depressive illness, to the related locomotive state and to various monoamingeric hypothesises that are suggested both in the genesis of affective disorders and the mechanism of action of two biochemically related depressive aspects, a distinction already considered by other authors for endogeneous depressions.