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.

Comparison of three 15N methods to correct for microbial contamination when assessing in situ protein degradability of fresh forages.

The use of stable (15)N as a marker to determine microbial contamination in nylon bag incubation residues to estimate protein degradability was investigated. Three methods using (15)N were compared: (15)N-labeled forage (dilution method, LF), (15)N enrichment of rumen solids-associated bacteria (SAB), and (15)N enrichment of rumen liquid-associated bacteria (LAB). Herbage from forages differing in protein and fiber contents (early-cut Italian ryegrass, late-cut Italian ryegrass, and red clover) were freeze-dried and ground and then incubated in situ in the rumen of 3 steers for 3, 6, 12, 24, and 48 h using the nylon bag technique. The (15)N-labeled forages were obtained by fertilizing the plots where herbage was grown with (15)NH4 (15)NO3. Unlabeled forages (obtained from plots fertilized with NH4NO3) were incubated at the same time that ((15)NH4)2SO4 was continuously infused into the rumen of the steers, and then pellets of labeled SAB and LAB were isolated by differential centrifugation of samples of ruminal contents. The proportion of bacterial N in the incubation residues increased from 0.09 and 0.45 g bacterial N/g total N at 3 h of incubation to 0.37 and 0.85 g bacterial N/g total N at 48 h of incubation for early-cut and late-cut ryegrass, respectively. There were differences (P < 0.001) between uncorrected N degradability values and those corrected for microbial contamination with all of the methods. Apparent N degradability of the low-N, high-fiber forage (late-cut ryegrass) was 0.51, whereas the corrected values were 0.85, 0.84, and 0.77 for the LF, SAB, and LAB methods, respectively. With early-cut ryegrass and red clover, the differences between uncorrected and corrected values ranged between 6% and 13%, with small differences among the labeling methods. Generally, methods using labeled forage or labeled SAB and LAB provided similar corrected degradability values. The accuracy in estimating the extent of degradation of protein in the rumen from in situ disappearance curves is improved when values are corrected for microbial contamination of the bag residue.

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.

Salmonella Typhimurium oral challenge model in mature broilers: bacteriological, immunological, and growth performance aspects.

In this study, Salmonella enterica serovar Typhimurium challenge models were tested to identify the best conditions under which to perform the experimental infection of 3-wk-old broilers. Such a model would be useful to study the efficiency of therapeutic treatments that could take place at the end of the grow-out period. Salmonella-free chicks were obtained from a breeder flock vaccinated with Salmonella. Intestinal maternal immunity was monitored by ELISA analyses at 2, 9, and 16 d of age. Data indicated that protection of maternal origin was not maintained over time and was drastically reduced at 9 d of age (P < 0.01). At 21 d of age, chickens were orally inoculated with Salmonella Typhimurium. The effects of the oral challenge dose (0, 3 × 10(3), 3 × 10(6), and 3 × 10(9) cfu/bird) and vancomycin pretreatment (no administration or 25 mg/bird) on intestinal immune responses, growth performance, and Salmonella colonization of chickens were investigated. After infection, the mucosal immune response was rapid, with increased (P < 0.01) anti-Salmonella Typhimurium IgA titers measured at 8 d postinfection in intestinal contents. A linear relationship (P < 0.05) existed between specific IgA levels in intestinal and cecal contents and the challenge dose inoculated. None of the challenge protocols caused mortality or clinical symptoms after infection. Nevertheless, the experimental infection induced a significant deterioration of growth performance. The pretreatment with 25 mg of vancomycin at 3 h before Salmonella inoculation was able to establish stable infection rates among the population of 3-wk-old infected chickens. Nevertheless, Salmonella shedding was not stable over the rearing period, and the bacteria seemed to be naturally eliminated from most birds at 22 d postinfection. This natural clearance of the gut, which was related, at least in part, to the intestinal immune response, should limit the usability of the created mature challenge model within 1 to 2 wk after inoculation.

Salmonella in chicken: current and developing strategies to reduce contamination at farm level.

Salmonella is a human pathogen that frequently infects poultry flocks. Consumption of raw or undercooked contaminated poultry products can induce acute gastroenteritis in humans. Faced with the public health concerns associated with salmonellosis, the European Union has established a European regulation forcing member states to implement control programs aimed at reducing Salmonella prevalence in poultry production, especially at the primary production level. The purpose of the present review article is to summarize the current research and to suggest future developments in the area of Salmonella control in poultry, which may be of value to the industry in the coming years. The review will focus especially on preventive strategies that have been developed and that aim at reducing the incidence of Salmonella colonization in broiler chickens at the farm level. In addition to the usual preventive hygienic measures, other strategies have been investigated, such as feed and drinking water acidification with organic acids and immune strategies based on passive and active immunity. Modification of the diet by changing ingredients and nutrient composition with the intent of reducing a bird's susceptibility to Salmonella infection also has been examined. Because in ovo feeding accelerates small intestine development and enhances epithelial cell function, this approach could be an efficient tool for controlling enteric pathogens. Feed additives such as antibiotics, prebiotics, probiotics, and synbiotics that modify the intestinal microflora are part of another field of investigation, and their success depends on the additive used. Other control methods such as the use of chlorate products and bacteriophages also are under study.

Effects of feed supplementation with specific hen egg yolk antibody (immunoglobin Y) on Salmonella species cecal colonization and growth performances of challenged broiler chickens.

Anti-Salmonella spp. egg yolk antibodies (IgY) simultaneously directed against Salmonella Enteritidis and Salmonella Typhimurium were tested to determine if their inclusion in feed decreased Salmonella spp. cecal colonization in experimentally infected broiler chickens. Egg yolk powder (EYP) was obtained by freeze-drying egg yolks containing anti-Salmonella spp. Immunoglobin Y was included in feed at 5 levels of concentration (0 to 5%). Feeds were formulated to similar nutrient levels and provided for ad libitum intake from d 1 to 28. Three days after initiation of feed treatments (d 4), chickens were co-challenged with equal numbers of Salmonella Enteritidis and Salmonella Typhimurium (2x10(6) cfu/bird). Cecal samples were recovered weekly over the experimental period (d 7 to 28) to enumerate Salmonella spp. The effect of anti-Salmonella spp. IgY feed supplementation on growth performance of infected chickens was also evaluated during the same period. In comparison with the positive control treatment (PC), treatments involving EYP (T1, T2, T3, T4, and T5), whether containing anti-Salmonella spp. IgY or not, significantly improved (P<0.05) the growth performance of challenged chickens, but without reaching the performance levels of nonchallenged chickens (NC1 and NC2). However, no link can be established between the enhancement in growth performance of challenged birds and their contamination levels by Salmonella because in-feed incorporation of EYP had no significant effect on cecal colonization by Salmonella. Furthermore, the comparison of the 5 anti-Salmonella spp. IgY concentration levels in feed did not reveal any anti-Salmonella spp. IgY concentration effect on growth performance and Salmonella cecal colonization. These results suggest that anti-Salmonella spp. IgY would undergo denaturation and degradation after their passage through the animal gastrointestinal tract and reveal that components of EYP other than specific antibodies have a beneficial effect on growth performance.

Efficiency of a Lactobacillus plantarum-xylanase combination on growth performances, microflora populations, and nutrient digestibilities of broilers infected with Salmonella Typhimurium.

Three experiments were performed to assess the ability of a Lactobacillus plantarum probiotic combined with a xylanase to reduce the effects of Salmonella Typhimurium infection in broiler chickens from 1 to 30 or 42 d of age. Chicks were challenged at 3 d of age with 10(8) or 10(5) cfu Salmonella Typhimurium/chick. Four diets were studied: a wheat-based diet (C+) supplemented with 0.1 g/kg of xylanase (E) or 10(6) cfu/g of L. plantarum (P), or both (PE). Uninfected chicks fed the C diet were used as negative control (C-). Six or 8 chicks were housed per cage with 9 cages/treatment. Growth performance and feed conversion ratio (FCR) were recorded weekly. In experiment 1, bacterial enumeration in ceca was achieved using the fluorescent in situ hybridization technique. Salmonella enumeration was realized in excreta by microbiological cultures (experiments 2 and 3). Nutrient digestibilities and AME(n) were determined in experiment 3 from d 35 to 39. Infection with Salmonella Typhimurium led to a significant decrease in the daily weight gain (DWG) by 23.6 to 32.8%, whereas FCR was increased by 1.0 to 19.7%. Chickens fed the PE diet showed significantly improved performance in comparison with C+ birds (DWG: +12.5% in experiment 1; FCR: -2.1 to 8.6%), and in comparison with the P and E treatments (DWG: +6.3 to 8.3% in experiment 1; FCR: -2.7 to 6.4%). In experiment 3, the FCR was significantly improved by 3% with the PE diet in comparison with C- chickens. The PE combination tended to restore a microflora similar to that of uninfected broilers, whereas the P and E diets had less of an effect on the profile of bacterial communities. At slaughter age, Salmonella contamination was reduced by 2.00 and 1.85 log colony-forming units for the E and PE treatment, respectively. The PE diet significantly reduced the crude fat digestibility by 9.2%, in comparison with the C+ chickens. These results suggest that the combination between L. plantarum and a xylanase as feed additive could be effective for reduction of the detrimental effect after Salmonella Typhimurium infection of broilers.

Effect of rumen-degradable protein balance deficit on voluntary intake, microbial protein synthesis, and nitrogen metabolism in growing double-muscled Belgian Blue bulls fed corn silage-based diet.

Two trials were conducted to evaluate the effect of rumen-degradable protein balance (OEB) deficit on voluntary intake (trial 1), microbial protein synthesis, and N metabolism (trial 2) in growing double-muscled Belgian Blue bulls. In trial 1, six bulls (339 +/- 26 kg of initial BW) were used in a replicated 3 x 3 Latin square and received a diet of 60% corn silage and 40% concentrate with ad libitum intake (DM basis). Three concentrates were formulated by adding urea at the expense of barley to give similar dietary contents of intestinal digestible proteins, NE for fattening, and fermentable OM, but with different levels of OEB. Thus, 2 levels of OEB deficit (-23.7 and -9.2 g of OEB/kg of DM) were compared with a diet providing a slight OEB surplus (5.3 g of OEB/kg of DM). Voluntary DMI decreased linearly (P = 0.02) with decreasing rumen-degradable protein balance. This decrease in intake could explain the linear decrease in ADG observed when negative OEB diets were fed. In trial 2, six bulls (304 +/- 12 kg of initial BW) with cannulas in the rumen and proximal duodenum were used in a replicated 3 x 3 Latin square and fed diets similar to those used in trial 1 at an intake level of 85 g of DM/kg of BW(0.75). Diurnal variations of ruminal NH(3)-N and plasma urea-N concentrations were greatly influenced by the level of OEB in the diet. No differences in NDF and starch degradation in the rumen, microbial N flow at the duodenum, or efficiency of microbial protein synthesis in the rumen were noted among the levels of OEB in diets. The reductions of the OEB value from 5.3 g/kg of DM to -9.2 g/ kg of DM and -23.7 g/kg of DM were associated with reductions of 26.5 and 48.8% in urinary N output. Absolute amounts of N retained by the bulls increased significantly with the level of OEB in diets. Indeed, 51.4% of the incremental supply of N was excreted between -23.7 and -9.2 g of OEB/kg of DM diets, and 74.6% of the incremental supply of N was excreted between -9.2 and 5.3 g of OEB/kg of DM diets. Feeding diets characterized by an adequate intestinal digestible protein supply and a OEB close to -10 g of OEB/kg of DM could be a feeding strategy to reduce N losses from the farm with little effect on the animal performance and voluntary intake. Reduced OEB may reduce N excretion in the environment but may also result in decreased N retention.

Production of hen egg yolk immunoglobulins simultaneously directed against Salmonella enteritidis and Salmonella typhimurium in the same egg yolk.

The present study was an attempt to raise hen egg yolk Ig (IgY) simultaneously directed against Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) in the same egg yolk. The immunopotentiating effect of 2 different adjuvants -- Freund's adjuvants (FA) and immunostimulating complexes matrix (IM) -- on antibody response was also evaluated. Bacterial outer membrane proteins (OMP) were selected as target antigens. The ISA Brown hens, specific-Salmonella spp.-free status, divided into 6 groups were intramuscularly injected with a mono-compound antigen preparation: SE-OMP (treatment SE-FA or SE-IM) or ST-OMP (treatment ST-FA or ST-IM), or a combined antigen preparation: (1/2) SE-OMP and (1/2) STOMP (treatment SEST-FA or SEST-IM). Titers of antibodies in yolk were evaluated biweekly with ELISA. There was no antigen x adjuvant interaction on antibody titers. Anti-SE IgY titers in hens that received treatment SEST-FA or SEST-IM were statistically similar (P > 0.05) as compared with those obtained from hens immunized with treatment SE-FA or SE-IM. Anti-ST IgY titers in hens immunized with treatment SEST-FA or SEST-IM were slightly lower than those of hens that received treatment ST-FA or ST-IM. The cross-reactivity of anti-SE IgY, induced by treatment SE-FA or SE-IM, with ST-OMP antigen and that of anti-ST IgY, induced by ST-FA or ST-IM, with SE-OMP antigen were arbitrarily assessed on d 43 and 155 by ELISA. The average cross-reactivity of anti-SE IgY with ST-OMP antigen was 71.7%. The average cross-reactivity of anti-ST IgY with SE-OMP antigen was 78.8%. In FA groups, antibody titers were found higher (P < 0.05) than those in IM groups. Furthermore, no extensive lesions or clinical abnormalities were detected in hens injected with FA. These findings showed the opportunity to raise IgY antibody against 2 Salmonella serovars in the same yolk and that FA was more efficient than IM in mediating antibody response.

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.

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.

Effect of various levels of imbalance between energy and nitrogen release in the rumen on microbial protein synthesis and nitrogen metabolism in growing double-muscled Belgian Blue bulls fed a corn silage-based diet.

Seven double-muscled Belgian Blue bulls (initial BW: 341 +/- 21 kg) with cannulas in the rumen and proximal duodenum were used in an incomplete replicated Latin square. The study examined the effect of imbalance between energy and N in the rumen on microbial protein synthesis and N metabolism by giving the same diet according to 3 different feeding patterns. The feed ingredients of the diet were separated into 2 groups supplying the same amount of fermentable OM (FOM) but characterized by different levels of ruminally degradable N (RDN). The first group primarily provided energy for the ruminal microbes (12.5 g of RDN/kg of FOM), whereas the second provided greater N (33.3 g of RDN/kg of FOM). These 2 groups were fed to the bulls in different combinations with the aim of creating 3 levels of imbalance (0, 20, and 40 g/ kg of DM) between energy and N supplies in the rumen. Imbalance was measured by the variation of the degradable protein balance (OEB value in the Dutch system) of the diet between the 2 meals each a day. Diurnal variations in ruminal NH3-N concentrations and plasma urea concentrations were greatly influenced by the feeding patterns of the diet. Introduction of imbalance affected neither microbial N flow at the duodenum (P = 0.97) nor efficiency of growth (P = 0.54). The feeding patterns of the diet had no negative impact on NDF degradation in the rumen (P = 0.33). Nitrogen retention was not affected by imbalance (P = 0.74) and reached 49.7, 52.0, and 51.3 g/d, respectively for 0, 20, and 40 g of OEB/kg of DM imbalance. It seems that introduction of an imbalance between energy and N supplies for the ruminal microbes by altering the feeding pattern of the same diet does not negatively influence the microbial activity in the rumen nor N retention of the animal. Nitrogen recycling in the rumen plays a major role in regulating the amount of ruminally available N and allows a continuous synchronization of N and energy-yielding substrates for the microorganisms in the rumen. Therefore, imbalance between dietary energy and N created over a 24-h interval was not detrimental to rumen microbial growth for the animal as long as the level of imbalance did not exceed 40 g of OEB/kg of DM. Thus, these feeding patterns of the diet can be used under practical feeding conditions with minimal impact on the performance of ruminant animals for meat production.

Effect of imbalance between energy and nitrogen supplies on microbial protein synthesis and nitrogen metabolism in growing double-muscled Belgian Blue bulls.

Six double-muscled Belgian Blue bulls (initial weight: 345 +/- 16 kg) with cannulas in the rumen and proximal duodenum were used in two juxtaposed 3 x 3 Latin squares to study the effect of a lack of synchronization between energy and N in the rumen on microbial protein synthesis and N metabolism by giving the same diet according to three different feeding patterns. The feed ingredients of the diet were separated into two groups supplying the same amount of fermentable OM (FOM), but characterized by different levels of ruminally degradable N (RDN). The first group primarily provided energy for the ruminal microbes (14.6 g of RDN/kg of FOM), and the second provided N (33.3 g of RDN/kg of FOM). These two groups were fed to the bulls simultaneously or alternately with the aim of creating three different time periods of imbalance (0, 12, or 24 h) between energy and N supplies in the rumen. The introduction of imbalance affected neither microbial-N flow at the duodenum (P = 0.65) nor efficiency of growth (P = 0.69), but decreased (P = 0.016) the NDF degradation in the rumen 12.2% for a 12-h period of imbalance. N retention was not affected by imbalance (P = 0.53) and reached 57.8, 58.5, and 54.7 g/d, respectively, for 0-, 12- and 24-h imbalance. It seems that the introduction of an imbalance of 12 or 24 h between energy and N supplies for the ruminal microbes by altering the feeding pattern of the same diet does not negatively influence microbial protein synthesis or N retention by the animal. Nitrogen recycling in the rumen plays a major role in regulating the amount ofruminally available N and allows for continuous synchronization of N- and energy-yielding substrates for the microorganisms in the rumen. Therefore, a lack of synchronization in the diet between the energy and N supplies for the ruminal microbes is not detrimental to their growth or for the animal as long as the nutrient supply is balanced on a 48-h basis. Thus, these dietary feeding patterns may be used under practical feeding conditions with minimal effect on the performance of ruminant animals.

Determination of the methionine requirement of growing double-muscled Belgian blue bulls with a three-step method.

The three-step technique was used to determine the requirements of total amino acids (TAA) and the first-limiting amino acid (AA) in growing double-muscled Belgian Blue bulls (BBb). In Exp. 1, three double-muscled BBb weighing initially 306 +/- 28 kg received a basal diet consisting of 30% meadow hay and 70% concentrate that was poor in digestible protein but had adequate NE because of continuous infusion of dextrose into the duodenum. The intestinal apparent digestibility of essential AA (EAA) was defined according to their duodenal and ileal flows. It averaged 72% but varied between 60% for Met and 79% for Arg. In Exp. 2, five double-muscled BBb (334 +/- 22 kg) received the same diet supplemented with duodenal infusions of dextrose and four doses of Na-caseinate (28, 56, 84, and 112% of intestinal digestible dietary AA) in a 4 x 4 Latin square design with one additional animal. Nitrogen retention for the basal diet alone and the four increasing supplements of Na-caseinate reached 49, 61, 70, 80, and 86 g/d, respectively. Nitrogen utilization improved from 34.3% without Na-caseinate supplementation to a maximum of 40.6%, with the third dose supplying 788 g/d of apparently digestible AA. Based on patterns of plasma concentrations, Met, Phe, and Arg were probably the limiting AA when animals optimized N utilization. In Exp. 3, six double-muscled BBb (315 +/- 25 kg) fed the basal diet received duodenal infusions of dextrose and AA, equivalent to the third dose in Exp. 2, except for digestible Met (9.3, 14.4, 18.4, 22.4, 26.4, and 30.4 g/d) in a 6 x 6 Latin square design. The Met requirement was close to 26.4 g/d on the basis of N retention.

The current 15N-leucine infusion technique is not suitable for quantitative measurements of ileal endogenous amino acid flows in pigs.

The current 15N-leucine infusion technique may overestimate the ileal endogenous nitrogen losses in pigs. To determine the reason, we infused four cannulated pigs intravenously, fed them a pea-based diet with 15N-leucine, and examined some methodological variables. Neither the blood sampling time nor the choice of precursor pool (total N or amino acid N of deproteinized plasma) or the method of estimation of the isotopic equilibrium level significantly affected the results. On the other hand, the 15N-enrichment of purified mucin, isolated from ileal digesta, was higher than that of the plasma amino acid pool (0.114 vs. 0.077 atom % excess). The endogenous proportion of the labeled amino acids (Ala, Gly, Ile, Leu and Val) in the ileal digesta ranged from 23 (Leu) to 74% (Ala), compared with 70% for total N. The low value of leucine was ascribed to the constant marker infusion condition. In pigs infused with 13C-leucine, a similar endogenous proportion was obtained for lumenal leucine with 13C-leucine and 15N-leucine infusion. However, the 13C-enrichment of the leucine bound to mucin was markedly lower than that of plasma leucine (38%). The endogenous amino acid flows were also estimated by combining the ileal N flow measured with 15N and the endogenous amino acid profile obtained by means of an N-free diet. They were different from those obtained with the 15N-amino acid dilution technique. We conclude that the precursor pool currently used (plasma total N or total alpha-amino acid N pools) is a poor indicator of the enrichment of the secretions and that the infusion of one labeled amino acid is not sufficient to extend the method at the amino acid level.

The use of 15N-labeled dietary proteins for determining true ileal amino acid digestibilities is limited by their rapid recycling in the endogenous secretions of pigs.

We assessed the use of 15N-labeled dietary proteins as a possible tool for the determination of the true ileal amino acid (AA) digestibility in pigs. The first experiment was designed to study the dietary N excretion pattern at the ileum subsequent to the ingestion of a single 15N-labeled meal. In a second experiment, we compared ileal endogenous AA outputs and true AA digestibility estimates obtained in pigs ingesting 15N-labeled dietary proteins in a single meal vs. intravenous infusion of [15N]leucine for 10 d during the ingestion of a pea-based diet and a protein-free starch diet. The proportion of endogenous N found in the ileal digesta differed when the label was delivered orally (50%) vs. intravenously (72%) and changed with time. As a consequence, the true ileal AA digestibilities measured with labeled diets were lower. A third experiment demonstrated that this was due to the rapid recycling of labeled dietary N in endogenous moieties, because 15N was found in blood within 10 min of consuming the labeled meal, within 50 min of consumption in pancreatic enzymes, 90 min in bile and 4 h in ileal mucins. We conclude that the use of 15N-labeled meals for determination of true ileal AA digestibilities is limited by the fast recycling of dietary N in endogenous secretions following a single 15N-labeled meal. The accuracy of results will depend on meaningful estimates of AA flow during a limited period and accurate estimates of 15N in AA.

Studies on the in situ nitrogen degradability corrected for bacterial contamination of concentrate feeds in steers.

Stable 15N was used to evaluate the influence of bacterial contamination on in situ DM and N degradabilities (Dg) of meat and bone meal (MBM), soybean meal (SBM), and wheat bran (WB) in two steers. Bacterial DM and N contamination ranged from 2.4 to 28.6% and 2.1 to 56.8% of residual DM and N, respectively. Effective N degradability increased when bacterial contamination was taken into account (P < .05). The difference was low for MBM (2.4%) and for SBM (3.4%) but high for WB (12.2%). Theoretically, using solid-associated bacteria should give the most accurate correction for bacterial contamination; however, results showed that Dg of N based on liquid-associated bacteria were identical for MBM and SBM (P > .05) and different for WB (P < .05). In a second experiment, five treatments were applied to incubated feeds to remove bacteria fixed to the residues and consequently to determine directly the Dg of DM and N corrected for the bacterial contamination without the need for a marker. These treatments involved chilling for 6 h at 4 degrees C in saline solution alone (T1) or with a commercial detergent (T2), or with sodium dodecyl sulfate (T3) or with methylcellulose (T4), followed by pummeling in a stomacher for 5 min. The last treatment was only machine washing twice (T5). The Dg of DM can be directly determined following the first four treatments, nevertheless their application to MBM and SBM led to higher Dg of N than that corrected for the bacterial contamination determined in the first experiment (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

[Influence of the conditioning method of a green forage on its chemical composition, the in vitro digestibility of the organic matter and the in sacco degradability of nitrogen in the rumen]. / Influence du mode de conditionnement d'un fourrage vert sur sa composition chimique, la digestibilité in vitro de la matière organique et la dégradabilité in sacco de l'azote dans le rumen.

Fiber contents of oven-dried ray-grass cut at different stages of maturity were higher than the values obtained for freeze-dried samples. The reverse was observed for in vitro organic matter digestibility. Moreover, oven-drying dramatically decreased in sacco nitrogen degradability as compared to lyophilization, preservation by freezing with liquid nitrogen and use of fresh material.