Effects of Saccharomyces cerevisiae, medium and forage type and their interactions on in vitro ruminal fermentation.

The objective of this study was to investigate the effects of a live yeast, Saccharomyces cerevisiae CNCM I-1077, at four doses (0, 1×105, 1×106 and 1 × 107 cfu/mL) according to the reducing medium used [Goering-Van Soest (GV), McDougall (MD) or Kansas State (KS)] on in vitro ruminal neutral detergent fibre digestibility (NDFd), rate of digestion of NDF (kd), organic matter digestibility (OMd), dry matter digestibility (DMd), pH as well as volatile fatty acids (VFA) concentration, using two forages (oat hay and wheat straw) with differing chemical composition. The maximum in vitro NDFd, DMd, OMd as well as kd were obtained with dose 1 × 106 cfu/mL, although differences between doses were not always significant. The pH estimates were the lowest with the 1 × 107 cfu/mL dose, but the differences were not all significant; however, 1 × 107 cfu/mL corresponded to significantly lower pH estimates compared to the control and 1×105 (6.51 vs. 6.60 and 6.59, respectively). The decrease in pH was accompanied by an increase in VFA concentrations as the yeast dose increased. The KS medium resulted in the lowest digestibility estimates, pH estimates as well as kd, regardless of yeast dose. The 1 × 106 cfu/mL was the better performing yeast dose in vitro resulting in higher digestibility estimates which indicates the yeasts ability to stimulate the microorganisms within the rumen by beneficially modifying rumen environment, thus promoting microbiota activity. The MD and GV media provide better environments for fermentation than the KS medium, resulting in higher in vitro NDFd, DMd, OMd, pH estimates as well as rate of NDF digestion. The MD and GV are also the media that resulted in more consistent results when analysing the effects of the live yeast. Our data suggest that the in vitro conditions have to be carefully chosen to be able to demonstrate rumen fermentation shifts with the use of live microbial additives.

Early supplementation of Saccharomyces cerevisiae boulardii CNCM I-1079 in newborn dairy calves increases IgA production in the intestine at 1 week of age.

The early development of immunity and microbiota in the gut of newborn calves can have life-long consequences. Gut microbiota and the intestinal barrier interplay after birth, establishing a homeostatic state whereby mucosal cells cohabit with microorganisms to develop a healthy gut. We hypothesized that postnatal codevelopment of gut immunity and microbiota could be influenced by early-life supplementation with live yeast. Starting from birth, calves either received a daily supplementation of Saccharomyces cerevisiae boulardii CNCM I-1079 (SCB, 10 × 109 cfu/d, n = 10) in the morning meal for 7 d or no supplementation (n = 10). Each animal received 2 adequate colostrum replacer meals at 2 and 12 h of life (expected total IgG fed = 300 g) before being fed milk replacer twice a day. Passive transfer of immunity (total protein, IgG, and IgA) through colostrum was evaluated and endogenous production of IgA was investigated by measuring IgA-producing plasma cells, IgA relative gene expression (PIGR and CD79A), and secretory IgA concentration in the gut. The concentration of targeted microbial groups was evaluated with quantitative PCR in the gut digesta collected at d 7 of life. Early SCB supplementation did not impair immunoglobulin absorption and all calves had successful passive transfer of immunity (serum IgG concentration >15 mg/mL at d 1 and d 7 of age). Although the expression of IgA relative gene expression (PIGR and CD79A) was not different, SCB calves had higher secretory IgA concentrations in the ileum (1.98 ± 0.12 mg/g of dry matter; DM) and colon (1.45 ± 0.12 mg/g of DM) digesta compared with control animals (1.18 and 0.59 ± 0.12 mg/g of DM, respectively). In addition, the number of IgA-producing plasma cells were greater in both ileum (2.55 ± 0.40 cells/mm2) and colon (3.03 ± 0.40 cells/mm2) tissues for SCB calves compared with control (respectively 1.00 ± 0.40 and 0.60 ± 0.42 cells/mm2). Endogenous IgA production in the gut of SCB calves was enhanced, which could make them less prone to pathogen intrusion. In addition, SCB calves had higher Lactobacillus and tended to have higher Faecalibacterium prausnitzii in the jejunum compared with control calves, which suggests that SCB supplementation during early-life gut colonization may have a positive effect in newborn calves. Direct SCB supplementation or the cross-talk between SCB and bacteria may be responsible for stimulating IgA production and may play a key role in shaping early colonization in the gut of newborn calves.

Changes in the rumen and colon microbiota and effects of live yeast dietary supplementation during the transition from the dry period to lactation of dairy cows.

The first objective of this study was to evaluate the dynamics and their potential association with animal performance of the microbiota in both the rumen and colon of dairy cows as they move from a nonlactation to a lactation ration. The second objective was to assess the potential effects on the microbiota of live yeast supplementation. Twenty-one Holstein cows were split in 2 treatments consisting of 1 × 1010 cfu/d of live yeast (LY; n = 10) or no supplementation (control; n = 11) starting 21 d before until 21 d after calving. At 14 d before and 7 and 21 d after calving, samples of rumen and colon digesta were obtained from each cow using an endoscope. Total DNA was extracted and submitted to high-throughput sequencing. Shannon diversity index, in both the rumen and colon, was unaffected by LY; however, in the rumen it was lowest 7 d after calving and returned to precalving values at 21 d in milk, whereas in the colon it was greatest 14 d before calving but decreased after calving. In the rumen, LY supplementation increased the relative abundance (RA) of Bacteroidales (group UCG-001), Lachnospiracea (groups UCG-002 and UCG-006), and Flexilinea 14 d before calving, and increased RA of Streptococcus 21 d after calving compared with control cows. However, changes in the ruminal microbiota were more drastic across days relative to calving than as influenced by the dietary treatment, and the effect of LY in the colon was milder than in the rumen. The ruminal RA of several genera was associated with postcalving DMI, and that of Gastranaerophilales was the only order positively associated with milk yield. Several genera were positively correlated with feed efficiency, with Clostridiales (unclassified) being the only genus negatively associated with feed efficiency. In the colon, Prevotellaceae (group Ga6A1) was the only genus positively associated with feed efficiency. The ruminal RA of Prevotella 7 and Ruminobacter 14 d precalving was negatively correlated with dry matter intake and milk yield postcalving. The RA of Parabacteroides in the colon 14 d before calving was negatively correlated with milk yield, whereas the RA of Eggerthellaceae (unclassified) and Erysipelotrichaceae (groups c and unclassified) were positively correlated with feed efficiency. Interestingly, LY supplementation doubled the RA of Eggerthellaceae (unclassified) in the colon. It is concluded that microbial diversity in the rumen experiences a transient reduction after calving, whereas in the colon, the reduction is maintained at least until 21 d in milk. Most of the effects of LY on rumen microbiota were observed before calving, whereas in the colon, LY effects were more moderate but consistent and independent of the stage of production. The microbial community of the rumen after calving is more associated with feed intake, milk yield, and feed efficiency than that of the colon. However, the colon microbiota before calving is more associated with feed efficiency after calving than that of the rumen.

Changes in gene expression in the rumen and colon epithelia during the dry period through lactation of dairy cows and effects of live yeast supplementation.

The objectives of this study were (1) to use endoscopy to collect biopsies from the rumen and colon epithelia to describe changes in gene expression in these 2 tissues as cows move from a dry to a lactation ration and (2) to evaluate the potential influence that supplementation of live yeast could exert on these 2 epithelia. Twenty-one Holstein cows were split into 2 treatments and received either 300 g/d of corn containing 1 × 1010 cfu/d of live yeast (LY; n = 10) or 300 g/d of corn with no supplementation (control; n = 11) starting 21 ± 2.6 d (average ± SD) before until 21 d after calving. At 14 ± 2.6 d before the expected calving date, and exactly at 7 and 21 d after calving, rumen and colon biopsies were obtained from each cow using an endoscope. Total RNA was extracted from rumen and colon tissues, and the expression of IL10, TNFA, TLR4, IL1B, PCNA, MKI67, SGLT1, BAX, CASP3, OCLN, CLDN4, HSPA1A, HSPB1, DEFB1, and MCT1 (the latter only in rumen samples) was quantified by quantitative PCR. Overall, fluctuations in expression of the selected genes in the colon between the 2 stages of production and the 2 treatments were smaller than those found in the rumen. In the rumen epithelium, expression of TLR4 and DEFB1 was greatest before calving, with LY cows having a greater expression of TLR4 than control cows. Similarly, expression of IL10 was greatest in LY cows before calving. Expression of TNFA in the rumen epithelium of control cows was lowest at 21 DIM but in LY cows was kept steady among production stages. The expression of PCNA and MKI67 in the rumen epithelium was greatest at 7 DIM, indicating a high proliferation rate of this epithelium after calving. In the colon mucosa, expression of TLR4 and DEFB1 was greater than in the rumen, and DEFB1 expression was greater in LY cows than in control cows. The use of an endoscope allowed us to study the dynamics of rumen epithelium adaptation to increased supply of concentrate after calving, consisting of increased epithelia remodeling, reduction of the TLR4, and increased IL10 expression. Furthermore, the rumen epithelium of dry cows responded rapidly to live yeast, with changes in the expression of genes involved in the immune response becoming evident after 7 d of exposure to yeast. The expression of genes related to the immune response (mainly TLR4 and DEFB1) in the colon mucosa was greater than in the rumen, and the expression of DEFB1 was further stimulated by live yeast. It is concluded that the use of an endoscope allows the study of gene expression patterns in the rumen and hindgut epithelia. We report marked changes in the rumen wall and more modest changes in the colon when transitioning from a dry to a lactation ration. Furthermore, supplementation of live yeast fostered and increased expression of genes regulating inflammation and epithelial barrier in the rumen, and in the colon it increased the expression of DFEB1 coding for an antimicrobial peptide.

Bioavailability of aflatoxin B1 and ochratoxin A, but not fumonisin B1 or deoxynivalenol, is increased in starch-induced low ruminal pH in nonlactating dairy cows.

High-production dairy and beef systems require diets rich in starch. This practice may induce ruminal acidosis and also increase exposure to mycotoxins because starches in starch-rich diets are the main vehicles of mycotoxin contamination. The aim of this study was to investigate the effects of low ruminal pH on the bioavailability of 4 major mycotoxins [i.e., aflatoxin B1 (AFB1), ochratoxin A (OTA), deoxynivalenol (DON), and fumonisin B1 (FB1)]. Eight nonlactating dairy cows fitted with rumen cannulas were used in a double crossover experiment. The trial was divided into 4 periods with 2 periods per crossover. Cows were divided into 2 groups receiving a low (15% dry matter basis) and high-starch diet (30.8%) with and without live yeast supplementation (1×1010 cfu per cow) in the first and second crossover, respectively. At the end of each period, cows received a single dose of mycotoxin-contaminated feed containing 0.05, 0.2, 0.24, and 0.56mg of AFB1, OTA, DON, and FB1 per kg of feed, respectively. The fecal and urinary excretion of mycotoxins and their metabolites was monitored for up to 48h postdosing. As expected, ruminal pH decreased in cows fed the high-starch diet. The high-starch diet increased the bioavailability of OTA and AFB1. Urinary excretion of OTA 24h after mycotoxin administration increased 3-fold in the high-starch diet, correlated with lower fecal excretion. Similarly, a decrease in fecal excretion of AFB1 was accompanied by an increase in urinary excretion of its major metabolite, aflatoxin M1, 48h after mycotoxin administration. In contrast to AFB1 and OTA, the bioavailability of DON and FB1 remained unchanged. Yeast supplementation had no effect on the excretion balance of these 2 mycotoxins. In conclusion, these results show that high-starch diets increased the bioavailability of OTA and AFB1, most probably through the lowering effect on ruminal pH. This greater bioavailability potentially increases the toxic effects of these mycotoxins.

Live yeasts enhance fibre degradation in the cow rumen through an increase in plant substrate colonization by fibrolytic bacteria and fungi.

AIMS: To monitor the effect of a live yeast additive on feedstuff colonization by targeted fibrolytic micro-organisms and fibre degradation in the cow rumen. METHODS AND RESULTS: Abundance of adhering fibrolytic bacteria and fungi on feedstuffs incubated in sacco in the cow rumen was quantified by qPCR and neutral detergent fibre (NDF) degradation was measured. Saccharomyces cerevisiae I-1077 (SC) increased the abundance of fibre-associated Fibrobacter succinogenes on wheat bran (WB) and that of Ruminococcus flavefaciens on alfalfa hay (AH) and wheat silage (WS). The greatest effect was observed on the abundance of Butyrivibrio fibrisolvens on AH and soya hulls (SH) (P < 0·001). Fungal biomass increased on AH, SH, WS and WB in the presence of SC. NDF degradation of AH and SH was improved (P < 0·05) with SC supplementation. CONCLUSIONS: Live yeasts enhanced microbial colonization of fibrous materials, the degree of enhancement depended on their nature and composition. As an effect on rumen pH was not likely to be solely involved, the underlying mechanisms could involve nutrient supply or oxygen scavenging by the live yeast cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Distribution of this microbial additive could be an interesting tool to increase fibre digestion in the rumen and thereby improve cow feed efficiency.

Effect of camelina oil or live yeasts (Saccharomyces cerevisiae) on ruminal methane production, rumen fermentation, and milk fatty acid composition in lactating cows fed grass silage diets.

The potential of dietary supplements of 2 live yeast strains (Saccharomyces cerevisiae) or camelina oil to lower ruminal methane (CH4) and carbon dioxide (CO2) production and the associated effects on animal performance, rumen fermentation, rumen microbial populations, nutrient metabolism, and milk fatty acid (FA) composition of cows fed grass silage-based diets were examined. Four Finnish Ayrshire cows (53±7 d in milk) fitted with rumen cannula were used in a 4×4 Latin square with four 42-d periods. Cows received a basal total mixed ration (control treatment) with a 50:50 forage-to-concentrate ratio [on a dry matter (DM) basis] containing grass silage, the same basal total mixed ration supplemented with 1 of 2 live yeasts, A or B, administered directly in the rumen at 10(10) cfu/d (treatments A and B), or supplements of 60g of camelina oil/kg of diet DM that replaced concentrate ingredients in the basal total mixed ration (treatment CO). Relative to the control, treatments A and B had no effects on DM intake, rumen fermentation, ruminal gas production, or apparent total-tract nutrient digestibility. In contrast, treatment CO lowered DM intake and ruminal CH4 and CO2 production, responses associated with numerical nonsignificant decreases in total-tract organic matter digestibility, but no alterations in rumen fermentation characteristics or changes in the total numbers of rumen bacteria, methanogens, protozoa, and fungi. Compared with the control, treatment CO decreased the yields of milk, milk fat, lactose, and protein. Relative to treatment B, treatment CO improved nitrogen utilization due to a lower crude protein intake. Treatment A had no influence on milk FA composition, whereas treatment B increased cis-9 10:1 and decreased 11-cyclohexyl 11:0 and 24:0 concentrations. Treatment CO decreased milk fat 8:0 to 16:0 and total saturated FA, and increased 18:0, 18:1, 18:2, conjugated linoleic acid, 18:3n-3, and trans FA concentrations. Decreases in ruminal CH4 production to treatment CO were related, at least in part to lowered DM intake, whereas treatments had no effect on ruminal CH4 emission intensity (g/kg of digestible organic matter intake or milk yield). Results indicated that live yeasts A and B had no influence on animal performance, ruminal gas production, rumen fermentation, or nutrient utilization in cows fed grass silage-based diets. Dietary supplements of camelina oil decreased ruminal CH4 and CO2 production, but also lowered the yields of milk and milk constituents due to an adverse effect on intake.

Repeated acidosis challenges and live yeast supplementation shape rumen microbiota and fermentations and modulate inflammatory status in sheep.

This study aimed to investigate the impact of repeated acidosis challenges (ACs) and the effect of live yeast supplementation (Saccharomyces cerevisiae I-1077, SC) on rumen fermentation, microbial ecosystem and inflammatory response. The experimental design involved two groups (SC, n=6; Control, n=6) of rumen fistulated wethers that were successively exposed to three ACs of 5 days each, preceded and followed by resting periods (RPs) of 23 days. AC diets consisted of 60% wheat-based concentrate and 40% hay, whereas RPs diets consisted of 20% concentrate and 80% hay. ACs induced changes in rumen fermentative parameters (pH, lactate and volatile fatty-acid concentrations and proportions) as well as in microbiota composition and diversity. The first challenge drove the fermentation pattern towards propionate. During successive challenges, rumen pH measures worsened in the control group and the fermentation profile was characterised by a higher butyrate proportion and changes in the microbiota. The first AC induced a strong release of rumen histamine and lipopolysaccharide that triggered the increase of acute-phase proteins in the plasma. This inflammatory status was maintained during all AC repetitions. Our study suggests that the response of sheep to an acidosis diet is greatly influenced by the feeding history of individuals. In live yeast-supplemented animals, the first AC was as drastic as in control sheep. However, during subsequent challenges, yeast supplementation contributed to stabilise fermentative parameters, promoted protozoal numbers and decreased lactate producing bacteria. At the systemic level, yeast helped normalising the inflammatory status of the animals.

Effect of transportation on fecal bacterial communities and fermentative activities in horses: impact of Saccharomyces cerevisiae CNCM I-1077 supplementation.

This study evaluated the effect of transportation on fecal bacterial communities and activities in horses with or without supplementation of live yeast and attempted to link those effects with changes in blood stress markers. Four mature horses were assigned to a crossover design and fed a basal diet (60:40 forage to concentrate; 1.45% BW on a DM basis), with or without supplementation, of 2 × 10(10) cfu/d of Saccharomyces cerevisiae CNCM I-1077. After a 14-d adaptation to dietary treatments, the 5-d experiment started 1 d before transportation (d -1). At d 0, horses were simultaneously transported in a truck for 2 h. Feces were sampled 4 h after the morning meal of concentrate at d -1, 0 (immediately after transportation), and 3 for enumeration of the main functional bacterial groups and determination of fermentative variables. Within each dietary treatment, feces were pooled before DNA extraction and molecular analysis of the bacterial communities, using temporal temperature gradient electrophoreses (TTGE). Blood samples were collected at the same time for determination of white blood cells (WBC) counts and glucose and total protein concentrations. Regardless of dietary treatment, the neutrophil to lymphocyte ratio increased during transportation (P < 0.01), indicating that horses were stressed. In both treatments, TTGE profiles were clearly different before and 3 d after transportation, and the percentage of similarity between profiles at d -1 and 3 was greater in supplemented horses compared with the controls. From d 0 to 3, the molar percentage of propionate increased and total concentration of VFA and the acetate + butyrate to propionate ratio decreased, regardless of dietary treatment (P < 0.01, P = 0.02, and P < 0.01, respectively), whereas pH decreased only in control horses (P = 0.03). Regardless of day of sampling, fecal concentrations of lactate-utilizing bacteria and cellulolytic bacteria were greater in supplemented horses than in control horses (P = 0.04 and 0.08, respectively). Our results indicate that transportation for 2 h disturbed the fecal bacterial ecosystem in horses that could increase the risk of triggering microbial dysbiosis on a longer term in the equine large intestine. Supplementing Saccharomyces cerevisiae CNCM I-1077 could help reduce the negative impact of transportation on the fecal bacterial ecosystem.

Enterohaemorrhagic Escherichia coli gains a competitive advantage by using ethanolamine as a nitrogen source in the bovine intestinal content.

The bovine gastrointestinal tract is the main reservoir for enterohaemorrhagic Escherichia coli (EHEC) responsible for food-borne infections. Characterization of nutrients that promote the carriage of these pathogens by the ruminant would help to develop ecological strategies to reduce their survival in the bovine gastrointestinal tract. In this study, we show for the first time that free ethanolamine (EA) constitutes a nitrogen source for the O157:H7 EHEC strain EDL933 in the bovine intestinal content because of induction of the eut (ethanolamine utilization) gene cluster. In contrast, the eut gene cluster is absent in the genome of most species constituting the mammalian gut microbiota. Furthermore, the eutB gene (encoding a subunit of the enzyme that catalyses the release of ammonia from EA) is poorly expressed in non-pathogenic E. coli. Accordingly, EA is consumed by EHEC but is poorly metabolized by endogenous microbiota of the bovine small intestine, including commensal E. coli. Interestingly, the capacity to utilize EA as a nitrogen source confers a growth advantage to E. coli O157:H7 when the bacteria enter the stationary growth phase. These data demonstrate that EHEC strains take advantage of a nitrogen source that is not consumed by the resident microbiota, and suggest that EA represents an ecological niche favouring EHEC persistence in the bovine intestine.

Probiotics in animal nutrition and health.

The use of probiotics for farm animals has increased considerably over the last 15 years. Probiotics are defined as live microorganisms which can confer a health benefit for the host when administered in appropriate and regular quantities. Once ingested, the probiotic microorganisms can modulate the balance and activities of the gastrointestinal microbiota, whose role is fundamental to gut homeostasis. It has been demonstrated that numerous factors, such as dietary and management constraints, can strongly affect the structure and activities of the gut microbial communities, leading to impaired health and performance in livestock animals. In this review, the most important benefits of yeast and bacterial probiotics upon the gastrointestinal microbial ecosystem in ruminants and monogastric animals (equines, pigs, poultry, fish) reported in the recent scientific literature are described, as well as their implications in terms of animal nutrition and health. Additional knowledge on the possible mechanisms of action is also provided.

Quantification by real-time PCR of cellulolytic bacteria in the rumen of sheep after supplementation of a forage diet with readily fermentable carbohydrates: effect of a yeast additive.

AIM: To examine the effect of concentrate and yeast additive on the number of cellulolytic bacteria in the rumen of sheep. METHODS AND RESULTS: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens were quantified using real-time PCR (targeting 16S rDNA) in parallel to cellulolytic flora enumeration with cultural techniques. Whatever the conditions tested, R. flavefaciens was slightly more abundant than F. succinogenes, with both species outnumbering R. albus. Before feeding, the shift from hay to hay plus concentrate diet had no effect on rumen pH and on the number of the three specie; while after feeding, the concentrate-supplemented diet induced a decrease (-1 log) of the number of the three species concomitant with the rumen acidification. Overall, the presence of the live yeast resulted in a significant increase (two- to fourfold) of the Ruminococci. CONCLUSION: The use of real-time PCR allowed us to show changes in the number of cellulolytic bacterial species in vivo in response to diet shift and additives that could not be as easily evidenced by classical microbial methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the understanding of the negative impact of readily fermentable carbohydrates on rumen cellulolysis and the beneficial effect of yeast on rumen fermentation.

Establishment of cellulolytic bacteria and development of fermentative activities in the rumen of gnotobiotically-reared lambs receiving the microbial additive Saccharomyces cerevisiae CNCM I-1077.

We studied the effects of a yeast additive used in ruminant nutrition on the establishment of cellulolytic bacteria, on plant cell wall degradation and on digestive functions in the rumen of gnotobiotically-reared lambs. Cellulolytic bacteria inoculated to the lambs tended to become established earlier in the presence of Saccharomyces cerevisiae CNCM I-1077 (SC). In addition, their population was maintained at a higher level, when the physico-chemical conditions of the biotope were altered. In these lambs, specific activities of fibrolytic enzymes were greater, and in sacco degradation of wheat straw tended to increase. In the presence of SC there was a decrease in ruminal ammonia concentration and a higher volatile fatty acid (VFA) concentration when lambs were 20 to 50 days old. These data suggest that this yeast strain may stimulate the development of cellulolytic microflora and enhance microbial activity in the rumen of young ruminants. Such activity could be beneficial in preventing microbial imbalance and a reduction of rumen function efficiency in the case of nutritional transitions. Further studies with conventional animals will soon be performed in order to verify these dings.