The influence of an increased cobalt supply on ruminal parameters and microbial vitamin B12 synthesis in the rumen of dairy cows

The aim of the study was to examine the effects of an elevated dietary cobalt supply to dairy cows on rumen fermentation parameters and microbial vitamin B12 synthesis in the rumen. Five lactating dairy cows fitted with a ruminal and a duodenal cannula were subsequently fed either a ration containing only the native cobalt content (0.17 mg Co/ kg DM) or a ration supplemented with cobalt sulphate (0.29 mg Co/kg DM). The pH-value, the ammonia concentration as well as the concentration and the molar proportions of short chain fatty acids in the rumen were not significantly influenced by feeding the ration with the higher cobalt content. While there was no difference in microbial protein flow, the cobalamin flow at the duodenum was significantly elevated in supplemented animals (3.67 +/- 0.69 vs. 8.63 +/- 2.22 mg B12/d). The efficiency of cobalt utilisation for ruminal vitamin B12 synthesis was calculated to be 7.1 +/- 1.3% for the unsupplemented and 9.5 +/- 2.4% for the supplemented ration. Further investigation has to prove if there are any benefits for cows resulting from the elevated cobalamin synthesis measured, caused by feeding higher amounts of dietary cobalt.     

Added dietary cobalt or vitamin B12, or injecting vitamin B12 does not improve performance or indicators of ketosis in pre- and post-partum Holstein-Friesian dairy cows

Vitamin B₁₂ is synthesised in the rumen from cobalt (Co) and has a major role in metabolism in the peri-paturient period, although few studies have evaluated the effect of the dietary inclusion of Co, vitamin B₁₂ or injecting vitamin B₁₂ on the metabolism, health and performance of high yielding dairy cows. A total of 56 Holstein-Friesian dairy cows received one of four treatments from 8 weeks before calving to 8 weeks post-calving: C, no added Co; DC, additional 0.2 mg Co/kg dry matter (DM); DB, additional 0.68 mg vitamin B₁₂/kg DM; IB, intra-muscular injection of vitamin B₁₂ to supply 0.71 mg/cow per day prepartum and 1.42 mg/cow per day post-partum. The basal and lactation rations both contained 0.21 mg Co/kg DM. Cows were weighed and condition scored at drying off, 4 weeks before calving, within 24 h of calving and at 2, 4 and 8 weeks post-calving, with blood samples collected at drying off, 2 weeks pre-calving, calving and 2, 4 and 8 weeks post-calving. Liver biopsy samples were collected from all animals at drying off and 4 weeks post-calving. Live weight changed with time, but there was no effect of treatment (P>0.05), whereas cows receiving IB had the lowest mean body condition score and DB the highest (P<0.05). There was no effect of treatment on post-partum DM intake, milk yield or milk fat concentration (P>0.05) with mean values of 21.6 kg/day, 39.6 kg/day and 40.4 g/kg, respectively. Cows receiving IB had a higher plasma vitamin B₁₂ concentration than those receiving any of the other treatments (P<0.001), but there was no effect (P>0.05) of treatment on homocysteine or succinate concentrations, although mean plasma methylmalonic acid concentrations were lower (P=0.019) for cows receiving IB than for Control cows. Plasma β-hydroxybutyrate concentrations increased sharply at calving followed by a decline, but there was no effect of treatment. Similarly, there was no effect (P>0.05) of treatment on plasma non-esterified fatty acids or glucose. Whole tract digestibility of DM and fibre measured at week 7 of lactation were similar between treatments, and there was little effect of treatment on the milk fatty acid profile except for C15:0, which was lower in cows receiving DC than IB (P<0.05). It is concluded that a basal dietary concentration of 0.21 mg Co/kg DM is sufficient to meet the requirements of high yielding dairy cows during the transition period, and there is little benefit from additional Co or vitamin B₁₂.     

Rubber seed oil and flaxseed oil supplementation alter digestion,ruminal fermentation and rumen fatty acid profile of dairy cows

Rubber seed oil (RO) that is rich in polyunsaturated fatty acids (FA) can improve milk production and milk FA profiles of dairy cows; however, the responses of digestion and ruminal fermentation to RO supplementation in vivo are still unknown. This experiment was conducted to investigate the effect of RO and flaxseed oil (FO) supplementation on nutrients digestibility, rumen fermentation parameters and rumen FA profile of dairy cows. Forty-eight mid-lactation Holstein dairy cows were randomly assigned to one of four treatments for 8 weeks, including basal diet (CON) or the basal dietary supplemented with 4% RO, 4% FO or 2% RO plus 2% FO on a DM basis. Compared with CON, dietary oil supplementation improved the total tract apparent digestibility of DM, neutral detergent fibre and ether extracts ( P < 0.05). Oil treatment groups had no effects on ruminal digesta pH value, ammonia N and microbial crude protein ( P > 0.05), whereas oil groups significantly changed the volatile fatty acid (VFA) profile by increasing the proportion of propionate whilst decreasing total VFA concentration, the proportion of acetate and the ratio of acetate to propionate ( P < 0.05). However, there were no differences in VFA proportions between the three oil groups (P > 0.05). In addition, dietary oil supplementation increased the total unsaturated FA proportion in the rumen by enhancing the proportion of trans-11 C18:1 vaccenic acid (VA), cis-9, trans-11 conjugated linoleic acid (CLA) and α-linolenic acid (ALA) ( P < 0.05). These results indicate that dietary supplementation with RO and FO could improve nutrients digestibility, ruminal fermentation and ruminal FA profile by enhancing the VA, cis-9, trans-11 CLA and ALA composition of lactating dairy cows. These findings provide a theoretical basis for the application of RO in livestock production.     

Effect of protein supplementation on ruminal parameters and microbial community fingerprint of Nellore steers fed tropical forages

In tropical regions, protein supplementation is a common practice in dairy and beef farming. However, the effect of highly degradable protein in ruminal fermentation and microbial community composition has not yet been investigated in a systematic manner. In this work, we aimed to investigate the impact of casein supplementation on volatile fatty acids (VFA) production, specific activity of deamination (SAD), ammonia concentration and bacterial and archaeal community composition. The experimental design was a 4×4 Latin square balanced for residual effects, with four animals (average initial weight of 280±10 kg) and four experimental periods, each with duration of 29 days. The diet comprised Tifton 85 (Cynodon sp.) hay with an average CP content of 9.8%, on a dry matter basis. Animals received basal forage (control) or infusions of pure casein (230 g) administered direct into the rumen, abomasum or divided (50 : 50 ratio) in the rumen/abomasum. There was no differences (P>0.05) in ruminal pH and microbial protein concentration between supplemented v. non-supplemented animals. However, in steers receiving ruminal infusion of casein the SAD and ruminal ammonia concentration increased 33% and 76%, respectively, compared with the control. The total concentration of VFA increased (P<0.05) in steers receiving rumen infusion of casein. SAD and the microbial protein concentration did not vary significantly among treatments during the feeding cycle, but mean SAD values were greater in steers supplemented in the rumen and rumen/abomasum. Ruminal ammonia concentration was positively correlated with SAD in animals receiving ruminal infusion of casein. Polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) analysis revealed low similarity between treatments, animals and time of sample collection. Richness analysis and determination of the Shannon–Wiener index indicated no differences (P>0.05) in species richness and diversity of γ-proteobacteria, firmicutes and archaea between non-supplemented Nellore steers and steers receiving casein supplementation in the rumen. However, species richness and the Shannon–Wiener index were lower (P<0.05) for the phylum bacteroidetes in steers supplemented with casein in the rumen compared with non-supplemented animals. Venn diagrams indicated that the number of unique bands varied considerably among individual animals and was usually higher in number for non-supplemented steers compared with supplemented animals. These results add new knowledge about the effects of ruminal and postruminal protein supplementation on metabolic activities of rumen microbes and the composition of bacterial and archaeal communities in the rumen of steers.     

Intake, rumen fermentation and nutrient flow to the omasum in beef cattle fed grass silage fortified with sucrose and/or supplemented with concentrate

The objective was to determine the relative effects of a specific increase in grass silage sucrose concentration, or a specific supplement of a starch-based concentrate, on rumen fermentation and nutrient supply to the omasum in beef cattle. Four ruminally cannulated Holstein–Friesian steers were fed grass silage only (G), G plus 3 kg concentrates/day (GC), G plus 90 g sucrose/kg dry matter (DM) (GS) and G plus 90 g sucrose/kg DM plus 3 kg concentrates/day (GCS) in a 4 × 4 Latin Square design experiment. Omasal flow was estimated using Co-EDTA, Yb-acetate and indigestible neutral detergent fibre (INDF) as digesta flow markers and purine bases as microbial markers. Concentrate supplementation reduced (P < 0.01) silage and increased (P < 0.001) total DM intake whereas sucrose had no effect. There was a sucrose × concentrate interaction (P < 0.05) for rumen pH whereby addition of sucrose to grass silage alone decreased pH and to grass silage plus concentrate had no effect. Rumen ammonia N (P < 0.01), total volatile fatty acid (VFA) concentration (P < 0.05) and the molar proportions of valerate (P < 0.05) and butyrate (P < 0.001) increased with concentrate supplementation whereas, sucrose supplementation had no effect on rumen fermentation parameters. Organic matter (OM) intake, omasal OM flow, the quantities of OM apparently (OMAD) and truly digested (OMTD) in the rumen (P < 0.001) and total tract OM digestibility (P < 0.01) increased, and apparent and true ruminal OM digestibility decreased (P < 0.05) with concentrate supplementation. Supplementation with concentrate decreased (P < 0.05) ruminal neutral detergent fibre (aNDFom) digestibility and increased (P < 0.05) aNDFom omasal flow. There was a tendency for addition of sucrose to increase (P < 0.1) ruminal OMAD and OMTD, while there was no effect of sucrose addition on intake or digestion of aNDFom. Concentrate supplementation increased (P < 0.001) N intake, flows of N, non-ammonia N (NAN), microbial N (MN) (P < 0.05) and non-ammonia non-microbial N (NANMN) (P < 0.01) and apparent total tract digestibility of N (P < 0.01), whereas sucrose reduced (P < 0.05) N intake and apparent ruminal N digestibility. There was no effect of concentrate or sucrose on N use efficiency or efficiency of microbial protein synthesis. Concentrate supplementation increased (P < 0.001) plasma β-hydroxybutyrate levels. In comparison to supplementing unwilted, well preserved grass silage of moderate digestibility with 3 kg starch-based concentrate per day, the limited response to the rate of sucrose supplementation employed suggests that increasing the water-soluble carbohydrate (WSC) concentration of grass silage through agronomic and/or ensiling practices will have relatively little effect on intake, rumen digestion or efficiency of microbial N synthesis.     

Effect of a controlled-release urea supplement on rumen fermentation in sheep fed a diet of sugar cane tops (Saccharum officinarum), corn stubble (Zea mays) and King grass (Pennisetum purpureum)

Four cannulated sheep were used to study ruminal fermentation of a diet consisting of 60% sugar cane tops (Saccharum officinarum), 30% corn stubble (Zea mays), 10% King grass (Pennisetum purpureum) and 0% (control), 10, 20 or 30% controlled-release urea supplement (CRUS) (diets 1, 2, 3 and 4, respectively). Average ruminal pH did not differ among diets (P>0.05), but during the first 6 h of sampling tended to be higher for CRUS diets. Ammonia concentrations were higher (P<0.01) in all treatments over controls, indicating microbial protein generation. Acetic acid production (mM/1) decreased (P<0.05), propionic acid increased (P<0.05), while butyric acid production did not differ among CRUS diets and controls (P>0.05). Total amounts of ruminal VFA were lowest (P<0.01) in controls, while CRUS diets produced more of these energy sources. Supplementation of the high fiber diets with 10, 20 or 30% CRUS increasingly improved rumen fermentation, ammonia supply and VFA production. The results show that low quality forages (up to 70% DMI) can be used efficiently by sheep when conditions for ruminal microorganism are improved with a controlled-release urea supplement.     

Effects of red cabbage extract rich in anthocyanins on rumen fermentation,rumen bacterial community,nutrient digestion,and plasma indices in beef bulls

Dietary anthocyanins (ATH) have probiotic and antioxidant functions in humans. They may also have beneficial impacts on rumen microorganisms and subsequently nutrient digestion in cattle. The experiment aimed to study the effects of dietary red cabbage extract (RCE) rich in ATH on rumen fermentation, rumen bacterial community, and nutrient digestibility in beef bulls. Eight Simmental beef bulls and two RCE levels (0 and 120 g/d) were allocated in a replicated 2 × 2 crossover design. Each experimental period included 15 days for adaptation and subsequent 5 days for sampling. The results showed that dietary addition of RCE increased the ruminal concentration of total volatile fatty acids and the molar proportion of propionate, decreased the acetate to propionate ratio, and tended to decrease the molar proportion of acetate, but it did not affect the ruminal pH and the concentrations of ammonia N, microbial CP, monophenols, polyphenols, and total phenolics. ATH was undetectable in the ruminal fluid of beef bulls in both groups. RCE did not affect the alpha diversity of rumen bacterial community, and the relative abundances of major rumen bacteria at the phylum level, but it increased the relative abundances of Ruminobacter and Anaerovibrio and tended to increase the relative abundances of Oribacterium and Monoglobus at the genus level. RCE tended to increase the plasma concentrations of globulin and total protein, but it did not affect the plasma albumin, urea, triglyceride, glucose, and antioxidant activities. Dietary addition of RCE did not affect the apparent nutrient digestibility. In conclusion, the ATH in RCE was highly hydrolysable in rumen fluid. Dietary addition of RCE increased the ruminal concentration of total volatile fatty acids, decreased the acetate to propionate ratio, and slightly modified the rumen bacterial community, but it did not affect the nutrient digestibility and the plasma antioxidants in beef bulls.     

Effects of isobutyrate supplementation in pre- and post-weaned dairy calves diet on growth performance,rumen development,blood metabolites and hormone secretion

Isobutyrate supplements could improve rumen development by increasing ruminal fermentation products, especially butyrate, and then promote the growth performance of calves. The objective of this study was to evaluate the effects of isobutyrate supplementation on growth performance, rumen development, blood metabolites and hormone secretion in pre- and post-weaned dairy calves. In total, 56 Chinese Holstein male calves with 30 days of age and 72.9±1.43 kg of BW, blocked by days of age and BW, were assigned to four groups in a randomized block design. The treatments were as follows: control, low-isobutyrate, moderate-isobutyrate and high-isobutyrate with 0, 0.03, 0.06 and 0.09 g isobutyrate/kg BW per calf per day, respectively. Supplemental isobutyrate was hand-mixed into milk of pre-weaned calves and the concentrate portion of post-weaned calves. The study consisted of 10 days of an adaptation period and a 50-day sampling period. Calves were weaned at 60 days of age. Seven calves were chosen from each treatment at random and slaughtered at 45 and 90 days of age. BW, dry matter (DM) intake and stomach weight were measured, samples of ruminal tissues and blood were determined. For pre- and post-weaned calves, DM intake and average daily gain increased linearly (P<0.05), but feed conversion ratio decreased linearly (P<0.05) with increasing isobutyrate supplementation. Total stomach weight and the ratio of rumen weight to total stomach weight tended to increase (P=0.073) for pre-weaned calves and increased linearly (P=0.021) for post-weaned calves, whereas the ratio of abomasum weight to total stomach weight was not affected for pre-weaned calves and decreased linearly (P<0.05) for post-weaned calves with increasing isobutyrate supplementation. Both length and width of rumen papillae tended to increase linearly for pre-weaned calves, but increased linearly (P<0.05) for post-weaned calves with increasing isobutyrate supplementation. The relative expression of messenger RNA for growth hormone (GH) receptor and 3-hydroxy-3-methylglutaryl-CoA synthase 1 in rumen mucosa increased linearly (P<0.05) for pre- and post-weaned calves with increasing isobutyrate supplementation. Blood concentrations of glucose, acetoacetate, β-hydroxybutyrate, GH and IGF-1 increased linearly (P<0.05) for pre- and post-weaned calves, whereas blood concentration of insulin decreased linearly with increasing isobutyrate supplementation. The present results indicated that isobutyrate promoted growth of calves by improving rumen development and its ketogenesis in a dose-dependent manner.     

Utilisation of fish oil in ruminants: I. Fish oil metabolism in sheep

Five sheep with rumen and abomasal cannulae were offered three diets sequentially in the order: control (C) pellets (lucerne hay-oat grain: 60/40, w/w), control plus unprotected tuna oil (UTO pellets), and control plus tuna oil protected (casein-formaldehyde matrix) against ruminal biohydrogenation (PTO pellets). In supplemented diets, tuna oil constituted 3% (w/w) of total dry matter (DM), and each supplement was fed for 12 days, with 9 days allowed between the two fish oil feeding periods to minimise carry-over effects. Daily DM intake was 785±38 g/head during the control period. It was significantly reduced by UTO feeding (6.2%, P<0.05) but not PTO feeding. The level of EPA in the abomasum during PTO feeding was double that measured during UTO feeding (1.30 versus 0.61% of FA, P<0.05). The level of DHA in the abomasum did not significantly differ between UTO and PTO feeding periods. Both tuna oil supplements significantly increased the levels of 18:1 trans and that of a fatty acid derivative identified as 10-hydroxystearic acid (10-HSA) in both the rumen and abomsum. Tuna oil supplementation also altered the fatty acid composition of plasma lipid fractions and 10-HSA was solely incorporated into plasma free fatty acids. This study indicates that substantial protection of tuna oil against ruminal hydrogenation inhibited reduced feed intake, but increased the proportion of 18:1 trans isomer and fatty acids derivatives (10-HSA), which indicate interference with metabolism in the rumen.     

Influence of barley grain particle size and treatment with citric acid on digestibility,ruminal fermentation and microbial protein synthesis in Holstein calves

Chemical and physical treatments of barley grain increase ruminally resistant starch and can improve the rumen fermentation pattern. The objective of the present study was to evaluate the effects of chemical (addition of citric acid, CA) and physical (grinding to two different particle sizes, PS) treatment of barley grain on performance, rumen fermentation, microbial protein yield in the rumen and selected blood metabolites in growing calves. In all, 28 male Holstein calves (172±5.1 kg initial BW) were used in a complete randomised design with a factorial arrangement of 2 barley grain particle sizes×2 levels of citric acid. The diets were as follows: (i) small PS (average 1200 µm) barley grain soaked in water (no CA addition); (ii) small PS barley grain soaked in a CA solution (adding 20 g CA/kg barley); (iii) large PS (average 2400 µm) barley grain soaked in water (no citric acid addition) and (iv) large PS barley grain soaked in a citric acid solution (adding 20 g CA/kg barley). Barley grain was then incorporated at 35% in a total mixed ration and fed to the calves for 11 weeks. Feeding small PS barley decreased feed intake (P=0.02) and average daily weight gain (P=0.01). The addition of CA to barley grain did not affect intake but increased weight gain (P<0.01) and improved feed to gain ratio (P=0.03). Digestibility of organic matter and NDF tended (P<0.10) to increase, whereas faecal scoring was improved (P=0.03) and the presence of undigested grain particles in faeces was reduced (P<0.01) with CA-treated barley grain. Glucose and urea concentrations were increased (P<0.01) in the blood of calves fed the CA-treated barley grain. Ruminal pH tended (P=0.08) to be decreased with more finely ground barley and was increased when barley grain was treated with CA. Total volatile fatty acid concentrations in the rumen did not differ among treatments (P>0.05). However, the molar proportion of propionate was increased (P=0.03) when barley was more finely ground, and that of acetate was increased (P=0.04) when CA was added to barley grain. The ruminal concentration of ammonia nitrogen was increased (P<0.01) and microbial nitrogen synthesis in the rumen tended to decrease by adding CA to barley. Treating barley grain with citric acid increased fibre digestibility of total mixed rations, attenuated the decrease in ruminal pH, and improved weight gain and feed efficiency in male Holstein growing calves fed a high-cereal diet (550 g cereal grain/kg diet).     

Garlic skin induces shifts in the rumen microbiome and metabolome of fattening lambs

Garlic (Allium sativum L.) and its constituents have been shown to modify rumen fermentation and improve growth performance. Garlic skin, a by-product of garlic processing, contains similar bioactive components as garlic bulb. This study aimed to investigate the effects of garlic skin supplementation on growth performance, ruminal microbes, and metabolites in ruminants. Twelve Hu lambs were randomly assigned to receive a basal diet (CON) or a basal diet supplemented with 80 g/kg DM of garlic skin (GAS). The experiment lasted for 10 weeks, with the first 2 weeks serving as the adaptation period. The results revealed that the average daily gain and volatile fatty acid concentration were higher (P < 0.05) in lambs fed GAS than those in the CON group. Garlic skin supplementation did not significantly (P > 0.10) affect the α-diversity indices, including the Chao1 index, the abundance-based coverage estimator value, and the Shannon and Simpson indices. At the genus level, garlic skin supplementation altered the ruminal bacterial composition by increasing (P < 0.05) the relative abundances of Prevotella, Bulleidia, Howardella, and Methanosphaera and decreasing (P < 0.05) the abundance of Fretibacterium. Concentrations of 139 metabolites significantly differed (P < 0.05) between the GAS and the CON groups. Among them, substrates for rumen microbial protein synthesis were enriched in the GAS group. The pathways of pyrimidine metabolism, purine metabolism, and vitamin B6 metabolism were influenced (P < 0.05) by garlic skin supplementation. Integrated correlation analysis also provided a link between the significantly altered rumen microbiota and metabolites. Thus, supplementation of garlic skin improved the growth performance of lambs by modifying rumen fermentation through shifts in the rumen microbiome and metabolome.     

Taxonomic Identification of Ruminal Epithelial Bacterial Diversity during Rumen Development in Goats

Understanding of the colonization process of epithelial bacteria attached to the rumen tissue during rumen development is very limited. Ruminal epithelial bacterial colonization is of great significance for the relationship between the microbiota and the host and can influence the early development and health of the host. MiSeq sequencing of 16S rRNA genes and quantitative real-time PCR (qPCR) were applied to characterize ruminal epithelial bacterial diversity during rumen development in this study. Seventeen goat kids were selected to reflect the no-rumination (0 and 7 days), transition (28 and 42 days), and rumination (70 days) phases of animal development. Alpha diversity indices (operational taxonomic unit [OTU] numbers, Chao estimate, and Shannon index) increased (P < 0.01) with age, and principal coordinate analysis (PCoA) revealed that the samples clustered together according to age group. Phylogenetic analysis revealed that Proteobacteria, Firmicutes, and Bacteroidetes were detected as the dominant phyla regardless of the age group, and the abundance of Proteobacteria declined quadratically with age (P < 0.001), while the abundances of Bacteroidetes (P = 0.088) and Firmicutes (P = 0.009) increased with age. At the genus level, Escherichia (80.79%) dominated at day zero, while Prevotella, Butyrivibrio, and Campylobacter surged (linearly; P < 0.01) in abundance at 42 and 70 days. qPCR showed that the total copy number of epithelial bacteria increased linearly (P = 0.013) with age. In addition, the abundances of the genera Butyrivibrio, Campylobacter, and Desulfobulbus were positively correlated with rumen weight, rumen papilla length, ruminal ammonia and total volatile fatty acid concentrations, and activities of carboxymethylcellulase (CMCase) and xylanase. Taking the data together, colonization by ruminal epithelial bacteria is age related (achieved at 2 months) and might participate in the anatomic and functional development of the rumen.     

Effects of Bacillus subtilis natto on milk production,rumen fermentation and ruminal microbiome of dairy cows

Two experiments were conducted to evaluate the effects of Bacillus subtilis natto, which was initially isolated from fermented soybeans on milk production, rumen fermentation and ruminal microbiome in dairy cows. In Experiment 1, 36 early lactation Chinese Holstein dairy cows (56 ± 23 days in milk) were randomly assigned to three groups: Control, cows were fed total mixed ration (TMR); BSNLOW, TMR plus 0.5 × 10¹¹ colony-forming units (cfu) of B. subtilis natto/cow per day; and BSNHIGH, TMR plus 1.0 × 10¹¹ cfu of B. subtilis natto/cow per day. During the 70-day treatment period, daily milk production and daily milk composition were determined in individual cows. The results showed that supplementing dairy cows with 0.5 × 10¹¹ and 1.0 × 10¹¹ cfu of B. subtilis natto linearly increased (P < 0.01) milk production (25.2 and 26.4 kg/day v. 23.0 kg/day), 4% fat-corrected milk (27.3 and 28.1 kg/day v. 24.2 kg/day), energy-corrected milk (27.3 and 28.2 kg/day v. 24.2 kg/day), as well as milk fat (1.01 and 1.03 kg/day v. 0.88 kg/day), protein (0.77 and 0.82 kg/day v. 0.69 kg/day) and lactose yield (1.16 and 1.22 kg/day v. 1.06 kg/day) but decreased milk somatic cell counts (SCC) by 3.4% to 5.5% (P < 0.01) in BSNLOW and BSNHIGH treatments compared with Control. In Experiment 2, four rumen-cannulated dairy cows were fed the basal diet from 1 to 7 days (pre-trial period) and rumen samples were collected on days 6 and 7; the same cows then were fed 1.0 × 10¹¹ cfu/day B. subtilis natto from days 8 to 21 (trial period) and rumen samples were collected on days 20 and 21. B. subtilis natto was discontinued from days 22 to 28 (post-trial period) and rumen samples were collected on days 27 and 28. Compared with the pre- and post-periods, ruminal pH decreased by 2.7% to 3.0% during the trial period (P < 0.01), whereas ammonia nitrogen (NH₃-N), total volatile fatty acids and molar proportion of propionate (P < 0.01) and valerate (P < 0.05) increased. Molar proportion of acetate decreased and the acetate to propionate ratio was lower (P < 0.01) during the trial period. However, no differences for 24-h in sacco dry matter digestibility were detected among different periods (treatments) though NDF digestibility was reduced in the trial and post-trial periods (P < 0.01). Compared with pre-trial period, total ruminal bacteria, proteolytic and amylolytic bacteria in rumen enumerated by culture methods increased by 15.0%, 16.2% and 11.7%, respectively (P < 0.01) but protozoa decreased to 5.35 log₁₀ cfu/ml (P < 0.01) during the trial period. These results demonstrate that B. subtilis natto improves milk production and milk components yield, decreases SCC and promotes the growth of total ruminal bacteria, proteolytic and amylolytic bacteria, which indicate that B. subtilis natto has potential to be applied as a probiotic for dairy cows.     

Effects of canola oil and antioxidants on performance,serum parameters,carcass traits,and rumen fermentation patterns of Nellore cattle

Several nutritional strategies have been used in beef cattle production in order to increase animal performance and profitability. However, in the past two decades, the increase of consumer preference for functional foods has driven the investigation for improving food via adding functional substances to animal diets. We evaluated the effect of canola oil supplementation associated with vitamin E and selenium on performance, rumen metabolism, carcass traits, meat tenderness, and serum, liver, and meat status of antioxidants in finishing Nellore males. Animals were fed for 106 days in a feedlot and were randomly distributed in a 2 × 2 factorial arrangement: two levels of oil in the diet (no inclusion and 3% canola oil, defined as diet without oil inclusion (NO) and effect of oil (OIL), respectively) and two levels of antioxidants in the diet (no inclusion and 2.5 mg of Se/kg of DM + 500 UI of vitamin E/kg of DM, defined as diet without antioxidant inclusion (NA) and effect of the antioxidants (ANT), respectively). DM intake (kg/day) was evaluated daily; performance and serum were analysed at the beginning of the feedlot and every 28 days. Animals were slaughtered and hot carcass weight (kg) was recorded; ruminal fluid and liver samples were collected. At 24 h postmortem, carcass pH was recorded and the Longissimus thoracis was sampled. There was no significant effect of the OIL*ANT interaction (P > 0.05) for any trait evaluated. Bulls fed OIL presented greater final BW (P < 0.01), average daily gain (kg/day; P < 0.01), feed efficiency (P < 0.01), rump fat thickness (P8RF; P < 0.05), and greater tenderness; the ANT diet increased P8RF (P < 0.05). The levels of selenium and vitamin E in serum, liver, and meat were increased (P < 0.01) with the inclusion of ANT. ANT did not change triiodothyronine (T3, ng/mL) and thyroxine (T4, µg/gL) serum concentrations but decreased serum glucose levels. The treatments did not affect (P > 0.05) ruminal parameters or the protozoa population. Our results showed that the inclusion of 3% canola oil in the diet DM increased performance, feed efficiency, carcass fat deposition, and tenderness, with no effect on rumen fermentation and protozoa population of Nellore cattle in a feedlot system. The inclusion of ANT in the cattle diet did not affect performance or rumen parameters. However, the levels of ANT were increased in the serum, liver, and meat, enriching the final product with these compounds.     

Quantitative analysis of ruminal bacterial populations involved in lipid metabolism in dairy cows fed different vegetable oils

Vegetable oils are used to increase energy density of dairy cow diets, although they can provoke changes in rumen bacteria populations and have repercussions on the biohydrogenation process. The aim of this study was to evaluate the effect of two sources of dietary lipids: soybean oil (SO, an unsaturated source) and hydrogenated palm oil (HPO, a saturated source) on bacterial populations and the fatty acid profile of ruminal digesta. Three non-lactating Holstein cows fitted with ruminal cannulae were used in a 3×3 Latin square design with three periods consisting of 21 days. Dietary treatments consisted of a basal diet (Control, no fat supplement) and the basal diet supplemented with SO (2.7% of dry matter (DM)) or HPO (2.7% of DM). Ruminal digesta pH, NH₃–N and volatile fatty acids were not affected by dietary treatments. Compared with control and HPO, total bacteria measured as copies of 16S ribosomal DNA/ml by quantitative PCR was decreased (P<0.05) by SO. Fibrobacter succinogenes, Butyrivibrio proteoclasticus and Anaerovibrio lipolytica loads were not affected by dietary treatments. In contrast, compared with control, load of Prevotella bryantii was increased (P<0.05) with HPO diet. Compared with control and SO, HPO decreased (P<0.05) C18:2 cis n-6 in ruminal digesta. Contents of C15:0 iso, C18:11 trans-11 and C18:2 cis-9, trans-11 were increased (P<0.05) in ruminal digesta by SO compared with control and HPO. In conclusion, supplementation of SO or HPO do not affect ruminal fermentation parameters, whereas HPO can increase load of ruminal P. bryantii. Also, results observed in our targeted bacteria may have depended on the saturation degree of dietary oils.     

The chemistry of vitamin B12. The coordination of biologically important molecules

The following equilibrium constants (given as logK in units of m⁻¹) were determined for the substitution of co-ordinated H₂O in aquocobalamin by glycine (bound through N) 5.8, cysteine (bound through S) 6.0 or 8.3, depending on the value chosen for the pK of the thiol group, and phenolate 2.9. The spectrum of the phenolate cobalamin shows an additional intense absorption band at 468nm with a molar extinction coefficient of 1.1×10⁴, which is assigned to a charge transfer from the phenolate to the cobalt ion. Equilibrium constants have also been determined for the equilibria between adenylcobamide cyanide and CN⁻, HO⁻ and H⁺, which show that the adenine is more easily displaced by CN⁻ and HO⁻ than is 5,6-dimethylbenziminazole in vitamin B₁₂, but can be protonated by acid while still remaining co-ordinated to the cobalt. It is shown that in the binding of corrinoids to proteins and polypeptides the formation of hydrogen bonds is far more important than co-ordination by the metal.     

Transient reductions in milk fat synthesis and their association with the ruminal and metabolic profile in dairy cows fed high-starch,low-fat diets

Sub-acute ruminal acidosis (SARA) is sometimes observed along with reduced milk fat synthesis. Inconsistent responses may be explained by dietary fat levels. Twelve ruminally cannulated cows were used in a Latin square design investigating the timing of metabolic and milk fat changes during Induction and Recovery from SARA by altering starch levels in low-fat diets. Treatments were (1) SARA Induction, (2) Recovery and (3) Control. Sub-acute ruminal acidosis was induced by feeding a diet containing 29.4% starch, 24.0% NDF and 2.8% fatty acids (FAs), whereas the Recovery and Control diets contained 19.9% starch, 31.0% NDF and 2.6% FA. Relative to Control, DM intake (DMI) and milk yield were higher in SARA from days 14 to 21 and from days 10 to 21, respectively (P < 0.05). Milk fat content was reduced from days 3 to 14 in SARA (P < 0.05) compared with Control, while greater protein and lactose contents were observed from days 14 to 21 and 3 to 21, respectively (P < 0.05). Milk fat yield was reduced by SARA on day 3 (P < 0.05), whereas both protein and lactose yields were higher on days 14 and 21 (P < 0.05). The ruminal acetate-to-propionate ratio was lower, and the concentrations of propionate and lactate were higher in the SARA treatment compared with Control on day 21 (P < 0.05). Plasma insulin increased during SARA, whereas plasma non-esterified fatty acids and milk β-hydroxybutyrate decreased (P < 0.05). Similarly to fat yield, the yield of milk preformed FA (>16C) was lower on day 3 (P < 0.05) and tended to be lower on day 7 in SARA cows (P < 0.10), whereas yield of de novo FA (<16C) was higher on day 21 (P < 0.01) in the SARA group relative to Control. The t10- to t11-18:1 ratio increased during the SARA Induction period (P < 0.05), but the concentration of t10-18:1 remained below 0.5% of milk fat, and t10,c12 conjugated linoleic acid remained below detection levels. Odd-chain FA increased, whereas branched-chain FA was reduced during SARA Induction from days 3 to 21 (P < 0.05). Sub-acute ruminal acidosis reduced milk fat synthesis transiently. Such reduction was not associated with ruminal biohydrogenation intermediates but rather with a transient reduction in supply of preformed FA. Subsequent rescue of milk fat synthesis may be associated with higher availability of substrates due to increased DMI during SARA.     

Effect of the supplementation of a high-concentrate diet with sunflower and fish oils on ruminal fermentation in sheep

This study was conducted to test the hypothesis that the supplementation of a high-concentrate diet with lipids, reportedly a good strategy for improving the nutritional value of ruminant-derived products, may not necessarily be associated with detrimental effects on ruminal fermentation in sheep. Four ruminally cannulated adult ewes were fed a high-concentrate diet, with no oil (Control diet), for a 14-day adaptation period. Afterwards, they were fed the same basal diet but supplemented with sunflower oil [20 g/kg fresh matter (FM)] and fish oil (10 g/kg FM) (SOFO diet) for a further 11 days, to investigate the impact of the addition of oils on the ruminal fermentation of the diet. On days 0 (Control), 3 and 10 of the experimental period rumen fluid was sampled at 0, 1.5, 3, 6 and 9 h after the morning feeding, for analysis of pH, and ammonia, lactate and total volatile fatty acid (VFA) concentrations. Alfalfa hay was incubated in situ, using the nylon bag technique, for 12 and 24 h to examine the effect of oil supplementation on ruminal disappearance of dry matter (DM), crude protein (CP) and neutral-detergent fibre (NDF). On days 0 and 11, rumen fluid was collected just before the morning feeding and used to incubate alfalfa hay and the Control and SOFO diets by means of the in vitro gas production technique. The mean concentrations of acetate (87.8 mmol/L vs. 73.7 mmol/L) and butyrate (21.2 mmol/L vs. 17.7 mmol/L) were reduced by oil supplementation (P < 0.05) and the total VFA showed a tendency (P = 0.098) to be lower with the SOFO diet (139.0 mmol/L vs. 122.1 mmol/L). However, none of the other in vivo ruminal fermentation parameters were affected by the treatment (P > 0.10). The oil supplementation affected neither in situ rumen disappearance of DM, CP and NDF of alfalfa hay, nor rates of gas production (P > 0.10). On the other hand, a little, but significant reduction in cumulative gas production was observed when the experimental diets were incubated with rumen fluid derived from animals fed the oil-rich diet (P < 0.05).Overall, the results suggest that the supplementation of high-concentrate diets with sunflower oil (20 g/kg FM) plus fish oil (10 g/kg FM) had little effect on ruminal fermentation and therefore its use to improve the nutritional value of ruminant-derived products cannot be precluded.     

Quantitative evaluation of ruminal methane and carbon dioxide formation from formate through C-13 stable isotope analysis in a batch culture system

Methane produced from formate is one of the important methanogensis pathways in the rumen. However, quantitative information of CH₄ production from formate has been rarely reported. The aim of this study was to characterize the conversion rate (CR) of formic acid into CH₄ and CO₂ by rumen microorganisms. Ground lucerne hay was incubated with buffered ruminal fluid for 6, 12, 24 and 48 h. Before the incubation, ¹³C-labeled H¹³COOH was also supplied into the incubation bottle at a dose of 0, 1.5, 2.2 or 2.9 mg/g of DM substrate. There were no interactions (P>0.05) between dose and incubation time for all variables evaluated. When expressed as an absolute amount (ml in gas sample) or a relative CR (%), both ¹³CH₄ and ¹³CO₂ production quadratically increased (P<0.01) with the addition of H¹³COOH. The total ¹³C (¹³CH₄ and ¹³CO₂) CR was also quadratically increased (P<0.01) when H¹³COOH was added. Moreover, formate addition linearly decreased (P<0.031) the concentrations of NH₃-N, total and individual volatile fatty acids (acetate, propionate and butyrate), and quadratically decreased (P<0.014) the populations of protozoa, total methanogens, Methanosphaera stadtmanae, Methanobrevibacter ruminantium M1, Methanobrevibacter smithii and Methanosarcina barkeri. In summary, formate affects ruminal fermentation and methanogenesis, as well as the rumen microbiome, in particular microorganisms which are directly or indirectly involved in ruminal methanogenesis. This study provides quantitative verification for the rapid dissimilation of formate into CH₄ and CO₂ by rumen microorganisms.     

Impact of oxalic acid on rumen function and bacterial community in sheep

Oxalic acid (OA) is a secondary compound occurring in a wide range of plants consumed by ruminants, especially in saline lands or in arid and semi-arid regions. However, its impact on the rumen microbial community and its changes over time, as well as the potential consequences on ruminal function, remain unknown. To examine this impact, five ewes fitted with a ruminal cannula and fed low-quality grass hay were dosed daily with 0.6 mmol of OA/kg body weight through the cannula for 14 days. On days 0 (before the start), 4, 7 and 14 of the administration period, samples of ruminal digesta were collected throughout the day (0, 3, 6 and 9 h after the morning feeding) for analysis of the bacterial community and fermentation parameters (pH, ammonia and volatile fatty acid (VFA) concentrations). In addition, two feedstuffs were incubated in situ using the nylon bag technique to estimate ruminal degradation. Terminal restriction fragment length polymorphism was employed to monitor the dynamics of total bacteria, and quantitative real-time PCR was used to investigate the abundance of the oxalate-degrading Oxalobacter formigenes. Neither pH nor total VFA concentrations were affected. Nevertheless, OA dosing altered molar proportions of most individual VFA and ammonia concentrations (P < 0.001). The dry matter disappearance of alfalfa hay was reduced on days 7 and 14 and that of barley straw only on day 7 (P < 0.01). These slight changes were related to others observed in the relative frequency of a number of terminal restriction fragments. Variations in the ruminal microbiota occurred rapidly with OA administration, which did not modify the bacterial diversity significantly but altered the structure of the community. However, many of these changes were reversed by the end of the experiment, with no significant differences between days 0 and 14 of dosing. These results suggest a rapid adaptation of the rumen bacterial community linked to the estimated increase in the abundance of O. formigenes (from 0.002% to 0.007% of oxc gene in relation to the total bacteria 16S rDNA; P < 0.01), which is assumed to be responsible for oxalate breakdown.