Effects of Dietary Supplementation With Clostridium butyricum on the Amelioration of Growth Performance, Rumen Fermentation, and Rumen Microbiota of Holstein Heifers

In China, the use of antibiotics growth promoters as feed additives has been banned. The goal of raising dairy heifers is to gain a relatively high body weight on a high-fiber diet at first mating or calving, thus increasing economic benefits. The objective of this experiment was to explore the effects of supplemental Clostridium butyricum (C. butyricum) on growth performance, rumen fermentation and microbiota, and blood parameters in Holstein heifers. Twenty Holstein heifers [mean ± standard deviation (SD); age = 182 ± 4.20 d, body weight = 197.53 ± 5.94 kg, dry matter intake (DMI) = 6.10 ± 0.38 kg] were randomly assigned to one of two diets group for a 42-day feeding period: (1) basal diet (an untreated control group, i.e., the CON group) or (2) basal diet plus daily 2 × 108 (colony-forming unit, CFU) of C. butyricum per kg of DMI per heifer (the CB group). The results demonstrated that C. butyricum supplementation increased the average daily gain from d 21 to 42 and DMI compared to the control group. Supplementation with C. butyricum significantly decreased the molar proportion of acetate and the acetate to propionate ratio but increased the molar proportion of butyrate and propionate. Compared with the control group, the relative abundance of Butyrivibrio fibrisolvens, Ruminococcus albus, Ruminobacter amylophilus, Ruminococcus flavefaciens, and Streptococcus bovis increased during the trial period in the CB group. However, C. butyricum had no significant effect on the blood parameters in Holstein heifers. In conclusion, these results show that feeding C. butyricum can improve growth performance and rumen fermentation without any negative impact on blood parameters in Holstein heifers.

Changes in Carbohydrate Composition in Fermented Total Mixed Ration and Its Effects on in vitro Methane Production and Microbiome

The purpose of this experiment was to investigate the changes of carbohydrate composition in fermented total mixed diet and its effects on rumen fermentation, methane production, and rumen microbiome in vitro. The concentrate-to-forage ratio of the total mixed ration (TMR) was 4:6, and TMR was ensiled with lactic acid bacteria and fibrolytic enzymes. The results showed that different TMRs had different carbohydrate compositions and subfractions, fermentation characteristics, and bacterial community diversity. After fermentation, the fermented total mixed ration (FTMR) group had lower contents of neutral detergent fiber, acid detergent fiber, starch, non-fibrous carbohydrates, and carbohydrates. In addition, lactic acid content and relative abundance of Lactobacillus in the FTMR group were higher. Compared with the TMR group, the in vitro ammonia nitrogen and total volatile fatty acid concentrations and the molar proportion of propionate and butyrate were increased in the FTMR group. However, the ruminal pH, molar proportion of acetate, and methane production were significantly decreased in the FTMR group. Notably, we found that the relative abundance of ruminal bacteria was higher in FTMR than in TMR samples, including Prevotella, Coprococcus, and Oscillospira. At the same time, we found that the diversity of methanogens in the FTMR group was lower than that in the TMR group. The relative abundance of Methanobrevibacter significantly decreased, while the relative abundances of Methanoplanus and vadinCA11 increased. The relative abundances of Entodinium and Pichia significantly decreased in the FTMR group compared with the TMR group. These results suggest that FTMR can be used as an environmentally cleaner technology in animal farming due to its ability to improve ruminal fermentation, modulate the rumen microbiome, and reduce methane emissions.

Tannic Acid-Steeped Corn Grain Modulates in vitro Ruminal Fermentation Pattern and Microbial Metabolic Pathways

This study investigated the effects of tannic acid (TA)-treated corn on changes in ruminal fermentation characteristics and the composition of the ruminal bacterial community in vitro. Ruminal fluid was obtained from three rumen-fistulated goats fed a 60:40 (forage/concentrate) diet. The batch cultures consisted of 25 ml of strained rumen fluid in 25 ml of an anaerobic buffer containing 0.56 g of ground corn, 0.24 g of soybean meal, 0.10 g of alfalfa, and 0.10 g of oat grass. Ground corn (2 mm) was steeped in an equal quantity (i.e., in a ratio of 1:1, w/v) of water alone (Con), 15 (TA15), 25 (TA25), and 35 g/l (TA35) TA solution for 12 h. After incubation for 24 h, TA-treated corn linearly increased (P <0.05) ruminal pH and the molar proportion of acetate, but linearly reduced (P <0.05) total volatile fatty acids and the molar proportion of butyrate compared with the Con treatment. Illumina MiSeq sequencing was used to investigate the profile changes of the ruminal microbes. A principal coordinates analysis plot based on weighted UniFrac values revealed that the structure of the ruminal bacterial communities in the control group was different from that of the TA-treated corn groups. The results of changes in the rumen bacterial communities showed that TA-treated corn linearly enriched (P <0.05) Rikenellaceae_RC9_gut_group, but linearly reduced (P <0.05) Ruminococcaceae_NK4A214_group, Ruminococcus_2, and unclassified_o__Clostridiales. Functional prediction of ruminal microbiota revealed that the TA-treated corn linearly decreased ruminal microbiota function of utilizing starch through pyruvate metabolism. In conclusion, TA-treated corn can modulate the rumen fermentation characteristics, microbial composition, and metabolic pathways, which may be potentially useful for preventing the occurrence of ruminal acidosis.

The rumen microbiome of yak co-evolves with its host probably adding the adaptation to its harsh environments

Background Rumen microbes play an important role in ruminant energy supply and animal performance. Previous studies showed that yak (Bos grunniens) rumen microbiome and fermentation differ from other ruminants. However, little is understood on the features of the rumen microbiome that make yak adapted to its unique environmental and dietary conditions. Here we investigated the rumen microbiome and metabolome to understand how yak adapts to the coarse forage and harsh environment in the high Qinghai-Tibetan plateau. Result Metataxonomic analysis of the rumen microbiota revealed that yak (Bos grunniens), domesticated cattle (Bos taurus), and dzo (a hybrid between the yak and domestic cattle) have distinct rumen microbiota. Metagenomic analysis displayed a larger gene pool encoding a richer repertoire of carbohydrate-active enzymes (CAZymes) in the rumen microbiome of yak and dzo than cattle. Some of the genes encoding glycoside hydrolases (GH) that mediate the digestion of cellulose and hemicellulose were significantly enriched in the rumen of yak than cattle, but the cattle rumen microbiome had more genes assigned to GH57 that primarily includes amylases. The rumen fermentation profile differed also, with cattle having a higher molar proportion of acetate but a lower molar proportion of propionate than dzo and yak. Metabolomic analysis showed differences in both rumen microbial metabolic pathways and metabolites, mainly amino acids, carboxylic acids, sugars, and bile acids. Notably, styrene degradation, primary bile acid biosynthesis, glyoxylate, and dicarboxylate metabolism significantly differed between cattle and dzo; streptomycin biosynthesis was significantly different between cattle and yak; and the pathways for biotin metabolism and styrene degradation significantly differed between dzo and yak. Correlation analysis revealed certain microbial species correlated with differential rumen metabolites. Nine differential metabolites showed a positive correlation with seven species belonging to Bacteroides and Alistipes but a negative correlation with ten species belonging to Prevotella and Ruminococcus. Conclusion The present study showed that the rumen microbiome of yak and its host had probably co-evolved aiding in the adaptation of yak to the harsh dietary environment of the Qinghai-Tibetan plateau. In particular, the yak rumen microbiome has more enzymes involved in the degradation of rough forage than that of cattle, providing sufficient energy for its host.

A Metagenomic Insight Into the Hindgut Microbiota and Their Metabolites for Dairy Goats Fed Different Rumen Degradable Starch

High starch diets have been proven to increase the risk of hindgut acidosis in high-yielding dairy animals. As an effective measurement of dietary carbohydrate for ruminants, studies on rumen degradable starch (RDS) and the effects on the gut microbiota diversity of carbohydrate-active enzymes (CAZymes), and Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology functional categories are helpful to understand the mechanisms between gut microbiota and carbohydrate metabolism in dairy goats. A total of 18 lactating goats (45.8 ± 1.54 kg) were randomly divided equally into three dietary treatments with low dietary RDS concentrations of 20.52% (LRDS), medium RDS of 22.15% (MRDS), and high RDS of 24.88% (HRDS) on a DM basis for 5 weeks. Compared with the LRDS and MRDS groups, HRDS increased acetate molar proportion in the cecum. For the HRDS group, the abundance of family Ruminococcaceae and genus Ruminococcaceae UCG-010 were significantly increased in the cecum. For the LRDS group, the butyrate molar proportion and the abundance of butyrate producer family Bacteroidale_S24-7, family Lachnospiraceae, and genus Bacteroidale_S24-7_group were significantly increased in the cecum. Based on the BugBase phenotypic prediction, the microbial oxidative stress tolerant and decreased potentially pathogenic in the LRDS group were increased in the cecum compared with the HRDS group. A metagenomic study on cecal bacteria revealed that dietary RDS level could affect carbohydrate metabolism by increasing the glycoside hydrolase 95 (GH95) family and cellulase enzyme (EC 3.2.1.4) in the HRDS group; increasing the GH13_20 family and isoamylase enzyme (EC 3.2.1.68) in the LRDS group. PROBIO probiotics database showed the relative gene abundance of cecal probiotics significantly decreased in the HRDS group. Furthermore, goats fed the HRDS diet had a lower protein expression of Muc2, and greater expression RNA of interleukin-1β and secretory immunoglobulin A in cecal mucosa than did goats fed the LRDS diet. Combined with the information from previous results from rumen, dietary RDS level altered the degradation position of carbohydrates in the gastrointestinal (GI) tract and increased the relative abundance of gene encoded enzymes degrading cellulose in the HRDS group in the cecum of dairy goats. This study revealed that the HRDS diet could bring disturbances to the microbial communities network containing taxa of the Lachnospiraceae and Ruminococcaceae and damage the mucus layer and inflammation in the cecum of dairy goats.

Effect of exogenous glucoamylase inclusion on in vitro fermentation and growth performance of feedlot steers fed a dry-rolled corn-based diet

The objective was to evaluate the effect of adding an exogenous glucoamylase (GA) enzyme from the fungus Trichoderma reesei on in vitro fermentation, growth performance, and carcass characteristics of feedlot steers fed a dry-rolled corn-based diet. Experiment 1 evaluated 3 levels of added enzyme (0, 0.24, and 0.72 GA enzyme units) and 2 corn particle sizes (CPS; 2 and 4 mm) in a factorial arrangement using a 7 h in vitro batch culture fermentation. Addition of GA increased (P < 0.01) in vitro dry matter disappearance by 13% and decreased final pH (P < 0.01). Molar proportion of propionate increased with GA inclusion (P < 0.01). A smaller CPS increased (P < 0.01) in vitro dry matter disappearance and total volatile fatty acid and decreased final pH (P < 0.01). A smaller CPS also decreased (P < 0.01) the molar proportion of acetate and increased (P < 0.01) the molar proportion of butyrate. In Experiment 2, Angus × Simmental steers (n = 105; initial BW = 329 ± 38 kg) were used to evaluate the inclusion of an exogenous GA on growth performance and carcass characteristics. Steers were fed a basal diet consisting of 60% dry-rolled corn, 17.5% modified distillers grains with solubles, 12.5% corn silage and 10% dry supplement on a dry matter basis for 136 d. Steers were blocked by weight and allotted to pens. Pens were randomly assigned to one of 3 treatments (5 pens/treatment): diet with no GA (CON), low inclusion of GA (122 enzyme units/kg DM; LGA), or high inclusion of GA (183 enzyme units/kg DM; HGA). Inclusion of GA did not affect (P ≥ 0.23) final BW, DMI, or ADG for the 136-d feeding period. Feed conversion was affected (P = 0.02) by treatment with steers fed HGA having ~8% greater G:F compared with LGA and CON. Treatment did not affect (P = 0.32) fecal starch. Inclusion of GA did not affect (P ≥ 0.19) carcass traits including HCW, 12 th rib fat thickness, yield grade, longissimus muscle area, or marbling score. Overall, results suggest inclusion of exogenous GA enzyme increased in vitro dry matter disappearance in batch culture and improved feed conversion in steers fed 183 enzyme units/kg DM during the finishing phase.

Effects of Calcium-ammonium Nitrate on in Vitro Ruminal Fermentation Using a Mature Mixed Winter Forage-based Substrate

A randomized complete block design was used to evaluate in vitro total gas production (TGP), methane production, concentration of NH3-N, and digestibility of mixed winter forage (CP 10.2% and NDF 58.6%) incubated with calcium-ammonium nitrate (CAN). In vitro fermentation consisted of 50 mL of a 4:1 buffer:ruminal fluid inoculum and 0.7 g of substrate [DM; wheat, triticale, and rye (Triticum aestivum, Triticosecale rimpaui, and Secale cereal; FOR) or forage:corn (90:10; CORN)] incubated for 48 h. Treatments included: 1) FOR; 2) CORN; 3) CORN + 2% CAN (DM; NIT); and 4) CORN + 0.67% UREA (DM; UREA). Treatments NIT and UREA were isonitrogenous. In vitro organic matter digestibility (IVOMD) was determined after incubation for 48 h, followed by a 48-h incubation with HCl and pepsin solutions. Data were analyzed using the MIXED procedure of SAS with the fixed effect of treatment and random effect of day (block). There was a treatment effect (P = 0.024) on TGP where NIT decreased TGP compared with CORN (P = 0.023), and NIT was not different from UREA. A greater IVOMD (P = 0.017) was observed for CORN compared with FOR. No differences were observed in IVOMD between UREA and NIT. There were no differences (P = 0.727) among all treatments for concentration of NH3-N. Total methane production was lesser for NIT (P ≤ 0.018) compared with all other treatments. There was a treatment effect for molar proportion of acetate (P = 0.039) and acetate:propionate (P = 0.034) where NIT tended (P = 0.058) to have a greater molar proportion of acetate compared to UREA. Total VFA concentration was not affected by treatment (P = 0.454). Calcium-ammonium nitrate influenced in vitro ruminal fermentation of a mature mixed winter forage, decreasing methane production without negatively affecting IVOMD or concentration of VFA.

Effects of bacterial cultures, enzymes and yeast-based feed additive combinations on ruminal fermentation in a dual-flow continuous culture system

Bacterial cultures, enzymes and yeast derived feed additives are often included in commercial dairy rations due to their effects on ruminal fermentation. However, the effects of these additives when fed together are not well understood. The objective of this study was to evaluate the changes in ruminal fermentation when a dairy ration is supplemented with combinations of bacterial probiotics, enzymes and yeast. Our hypotheses were that ruminal fermentation would be altered, indicated through changes in volatile fatty acid profile and nutrient digestibility, with inclusion of: (1) an additive, (2) yeast and (3) increasing additive doses. Treatments were randomly assigned to 8 fermenters in a replicated 4 × 4 Latin square with four 10 d experimental periods, consisting of 7 d for diet adaptation and 3 d for sample collection. Basal diets contained 52:48 forage:concentrate and fermenters were fed 106 g of dry matter per day divided equally between 2 feeding times. Treatments were: control (CTRL, without additives); bacterial culture/enzyme blend (EB, 1.7 mg per day); bacterial culture/enzyme blend with a blend of live yeast and yeast culture (EBY, 49.76 mg per day); and double dose of the EBY treatment (2X, 99.53 mg per day). The bacterial culture/enzyme blend contained five strains of probiotics (Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus lichenformis, Bacillus subtillis, and Enterococcus faecium) and three enzymes (amylase, hemicellulase, and xylanase). On d 8-10, samples were collected for pH, redox, volatile fatty acids, lactate, ammonia N and digestibility measurements. Statistical analysis was performed using the GLIMMIX procedure of SAS. Repeated measures were used for pH, redox, VFA, NH3-N and lactate kinetics data. Orthogonal contrasts were used to test the effect of: (1) additives, ADD (CTRL vs EB, EBY and 2X); (2) yeast, YEAST (EB vs EBY and 2X); and (3) dose, DOSE (EBY vs 2X). No effects (p > 0.05) were observed for pH, redox, NH3-N, acetate, isobutyrate, valerate, total VFA, acetate:propionate, nutrient digestibility or N utilization. Within the 24h pool, the molar proportion of butyrate increased (p = 0.03) with the inclusion of additives when compared to the control while the molar proportion of propionate tended to decrease (p = 0.07). In conclusion, inclusion of bacterial cultures, enzymes and yeast to the diet increased butyrate concentration; but did not result in major changes in ruminal fermentation.

Effects of calcium-magnesium carbonate and calcium-magnesium hydroxide as supplemental sources of magnesium on microbial fermentation in a dual-flow continuous culture

Supplemental sources of Mg can also aid in ruminal pH regulation due to their alkaline properties. Magnesium oxide (MgO) is the most common source of Mg for ruminants and can help controlling ruminal pH; however, alkaline potential of other sources of Mg has not been evaluated. We aimed to evaluate the inclusion of calcium-magnesium carbonate (CaMg(CO3)2) and calcium-magnesium hydroxide (CaMg(OH)4) alone or in combination as supplemental sources of Mg in corn silage-based diets and its impact on ruminal microbial fermentation. We hypothesized that inclusion of CaMg(OH)4 would allow for ruminal fermentation conditions resulting in a greater pH compared to inclusion of CaMg(CO3)2. Four treatments were defined by the supplemental source of Mg in the diet: 1) Control (100% MgO, plus sodium sesquicarbonate as a buffer); 2) CO3 [100% CaMg(CO3)2]; 3) OH [100% CaMg(OH)4]; and 4) CO3/OH [50% Mg from CaMg(CO3)2, 50% Mg from CaMg(OH)4]. Nutrient concentration was held constant across treatments (16% CP, 30% NDF, 1.66 MCal NEl/kg, 0.67% Ca, and 0.21% Mg). Four fermenters were used in a 4x4 Latin Square design with 4 periods of 10 d each. Samples were collected for analyses of nutrient digestibility, soluble Mg, VFA, and NH3, while pH was measured at 0, 1, 2, 4, 6, 8, and 10 h post morning feeding to estimate % time when pH was below 6 (pH-B6) and area under the pH curve for pH below 6.0 (pH-AUC). Bacteria pellets were harvested for 15N analysis and estimates of N metabolism. Treatment effects were analyzed with the mixed procedure of SAS, while effects of using either CaMg(CO3)2 or CaMg(OH)4 as Mg source in comparison to Control treatment were evaluated by orthogonal contrasts. Similar pH-related variables were observed for Control, OH, and CO3/OH treatments, which had smaller pH-AUC and pH-B6 than CO3 (P ≤ 0.01). Butyrate molar proportion was greater in Control and CO3/OH than in CO3 and OH (P = 0.04). Orthogonal contrasts showed lower flow of bacterial N (P = 0.04), lower butyrate molar proportion (P = 0.08) and greater pH-AUC (P = 0.05) for diets with CaMg(CO3)2 in comparison with the Control. Concentration of soluble Mg in ruminal fluid (P = 0.73) and nutrient digestibility (P ≥ 0.52) were similar across treatments. Under the conditions of this experiment, using CaMg(OH)4 alone or combined with CaMg(CO3)2 allowed for a less acidic ruminal fermentation pattern than a diet with only CaMg(CO3)2.

183 A blend of plant extracts (Actifor Pro) potentially reduced methane production in a dual-flow continuous culture system fed bermudagrass hay with or without corn gluten feed

Twelve dual-flow continuous culture fermenters (1.95 L) were used to evaluate the effect of a phytogenic feed additive (Actifor Pro, Delacon, Engerwitzdorf, Austria; ACT) on ruminal fermentation and methane production when fed high fiber diets, comprised of bermudagrass hay (BGH) with or without corn gluten feed (CGF). Fermenters were utilized in a generalized randomized block design with a 2 × 2 factorial arrangement of treatments (n = 6): 1) diet (with or without CGF at 22% of diet DM) and 2) additive (with or without ACT at 1.0 g/L ACT). Two 10-d periods were conducted. Overall, comparing to no CGF, supplementation with CGF resulted in lower dry matter, organic matter, crude protein digestibility, microbial efficiency of nitrogen utilization, and methane production (mL of CH4/mol of total VFA), but increased neutral detergent fiber digestibility (all P ≤ 0.05). Diet × additive interactions were observed for molar proportion of acetate and propionate, and acetate-to-propionate ratio (A:P, all interactions P < 0.05), where inclusion of ACT increased acetate molar proportion and A:P (both P < 0.05), and decreased propionate molar proportion in diets with CGF (P = 0.05). A diet × additive interaction was also observed for methane production (ml of CH4/mol of total VFA; P = 0.08), where ACT decreased CH4 production per mol of VFA by 42% (P = 0.04), when only BGH was fed to the fermenters. In conclusion, CGF supplementation in BGH diets reduced methane production and improved fiber digestibility. The decrease in methane production per mol of VFA observed with ACT in the BGH without CGF diet warrants further investigation.