Rumen Fluid Contents Research Articles

The Effect of Adding Green and Black Tea Waste Extracts on Rumen Fermentation Parameters by In Vitro Techniques

The increase in global temperatures over the past few decades due to greenhouse gas emissions has raised concerns and necessitated further research in climate change mitigation and adaptation. Methane is a prominent greenhouse gas that significantly contributes to climate change, with a substantial amount generated through fermentation processes occurring in the rumen of ruminant animals. The potential of plant secondary metabolites, especially those derived from tannin-rich plants, warrants investigation to modify rumen fermentation and mitigate methane emissions in livestock diets. The objective of this study was to assess the impact of extracts obtained from green and black tea waste on rumen fermentation dynamics and gas (methane) production, utilizing in vitro methods. For this purpose, rumen fluid was collected from two fistulated sheep and subjected to three treatments: (1) a basal diet (control), (2) a basal diet + green tea waste extract (5% of dry matter), (3) a basal diet + black tea waste extract (5% of dry matter). The study assessed the effects of incorporating extracts from green and black tea waste on various parameters, including digestibility, protozoa population, ammonia nitrogen levels, volatile fatty acids, and methane gas production following a 24-h incubation period. Statistical analysis of the data was conducted using SAS software within a completely randomized design framework. The findings indicated that the addition of green and black tea waste extracts significantly decreased methane gas production (p < 0.05), protozoa count (p < 0.05), and ammonia nitrogen concentrations in rumen fluid (p < 0.05) when compared to the control group. The addition of green and black tea waste extracts has significantly altered the concentration of VFAs in rumen fluid (p < 0.05). Specifically, the addition of green tea waste extract has led to a highly significant reduction in acetic acid, (p < 0.01) and the addition of both extracts has resulted in a significant increase in propionic acid (p < 0.05). Consequently, the results suggest that the inclusion of green and black tea waste extracts in livestock diets may effectively mitigate methane emissions in the rumen, thereby reducing feed costs and reducing environmental pollution.

Changes in the rumen microbial community composition of dairy cows subjected to an acidogenic diet

In modern breeding systems, cows are subjected to many stress factors. Animals fed with a high-grain diet may have a decreased rumen pH, which would lead to subacute ruminal acidosis syndrome. The aim of this study was to investigate the evolution of microbial community composition in cows undergoing a dietary stress challenge. Twelve cows were subjected to a challenge period consisted in a rapid change of ration, from a normal (45.4:54.6 forage: concentrate) to a high-grain content diet (24.8:75.2 forage: concentrate) to induce sub-acute ruminal acidosis. Individual rumen fluid content samples were collected before (T0), and during the challenge (T3, T14, T28). DNA from rumen contents was extracted, purified, and sequenced to evaluate Bacterial populations and sequencing was performed on Illumina MiSeq. The effect of animal conditions on rumen microbial community was quantified through a linear mixed model. The acidogenic diet created 2 main clusters: ruminal hypomotility (RH) and milk fat depression (MFD). The microbial composition did not differ in T0 between the 2 groups, while during the challenge Ruminococcus spp., Treponema spp., Methanobrevibacter spp., and Methanosphaera spp. concentrations increased in RH cows; Succinivibrio spp. and Butyrivibrio spp. concentrations increased in MFD cows. Prevotella spp. and Ruminococcus spp., were negatively correlated, while Christenellaceae family were positively correlated with both Methanobrevibacter spp. and Methanosphaera spp. Moreover, the same diet affected differently cows' microbiota composition, underlying the impact of the host effect. Other studies are necessary to deepen the relationship between microbiota composition and host.

The acute effects of rumen pulse-dosing of hydrogen acceptors during methane inhibition with nitrate or 3-nitrooxypropanol in dairy cows

Dietary methane (CH4) mitigation is in some cases associated with an increased hydrogen (H2) emission. The objective of the present study was to investigate the acute and short-term effects of acceptors for H2 (fumaric acid, acrylic acid or phloroglucinol) supplemented via pulse-dosing to dairy cows fed CH4 mitigating diets (using nitrate or 3-nitrooxypropanol), on gas exchange, rumen gas and VFA composition. For this purpose, 2 individual 4 × 4 Latin square experiments were conducted with 4 periods of 3 d (nitrate supplementation) and 7 d (3-nitrooxypropanol supplementation), respectively. In each study, 4 rumen cannulated Danish Holstein cows were used. Each additive for CH4 mitigation was included in the ad libitum fed diet within the 2 experiments, to which the cows were adapted for at least 14 d. Acceptors for H2 were administered twice daily in equal portions through the rumen fistula immediately after feeding of the individual cow. In Exp. 1 (nitrate), the treatments were CON-1 (no H2-acceptor), FUM-1 (fumaric acid), ACR-1 (acrylic acid) and FUM+ACR-1 (50% FUM-1 + 50% ACR-1). In Exp. 2 (3-nitrooxypropanol), the 3 treatments, CON-2, FUM-2, and ACR-2, were similar to CON-1, FUM-1 and ACR-1 treatments, however the fourth treatment was PHL-2 (phloroglucinol). Gas exchanges were measured in respiration chambers, while samples of rumen liquid and headspace gas were taken in time series relative to feeding and dosing on specific days. Headspace gas was analyzed for gas composition and rumen liquid was analyzed for volatile fatty acid composition and dissolved gas concentrations. Headspace gas composition and dissolved gas concentration were only measured in Exp. 2. Dry matter intake was reduced upon acrylic acid supplementation. There were no significant effects of any treatments in any experiments on H2 emission, except for a decrease in hourly H2 emission rate (g/h) at 1 h after feeding in both experiments. In Exp. 2, H2 headspace proportions increased by ACR-2 supplementation, whereas dissolved concentrations were unaffected. In Exp. 1, cows on ACR-1 increased propionate proportion at 1 h after feeding. In Exp. 2, both FUM-2 and ACR-2 increased rumen propionate proportion in the hours after feeding and dosing. There was no effect on rumen acetate for cows on PHL-2. There was a strong positive correlation between rumen dissolved CH4 and headspace CH4 (r = 0.84), whereas the equivalent correlation was weaker for H2 (r = 0.41). For the relationship between dissolved concentrations and emissions of CH4 and of H2, there was a moderate positive correlation for CH4 (r = 0.54), whereas it was weak for H2 (r = 0.28) with zero slope. In conclusion, the results suggested that fumaric acid and acrylic acid to some extent was reduced to propionate without associative effects on measures for H2 redirection. Furthermore, phloroglucinol seemed not to be metabolized in the rumen in the present study, as no effects on rumen acetate or measures of H2 were observed. Changes in H2 headspace and emission may be a poor proxy for actual changes in the rumen fluid concentration of H2.

85 Evaluation of a Saccharomyces cerevisiae fermentation product on performance, plasma metabolites, rumen parameters, and fecal pathogen shedding in feedlot steers

Abstract The objective of the experiment was to evaluate a commercial Saccharomyces cerevisiae fermentation product (SCFP; NaturSafe, Dimond V, Cedar Rapids, IA) on performance, plasma metabolites, rumen parameters, and fecal pathogen shedding in feedlot steers. Crossbred steers (n = 61; 271 ± 48.1 kg) were sourced from 3 different research units at Oklahoma State University and assigned to 1 of 3 experimental treatments in a randomized complete block design. Steers were group housed in pens equipped with an automated individual feed intake system. Treatments consisted of a negative control (CON), CON with tylosin phosphate (TYL; 9.9 mg · kg-1 of diet DM), or CON with SCFP (1.63 g · kg-1 of diet DM). Body weight and feed efficiency did not differ among treatments (P ≥ 0.12). However, steers fed SCFP tended to have greater ADG from d 35 to 56 (P = 0.10). In addition, steers fed SCFP exhibited increased DMI from d 56 to 112 (P = 0.03), a tendency for increased DMI from d 112 to final (P = 0.07), and increased DMI overall (P = 0.05). There was no treatment × time interaction for pH, pH change, or lactate concentrations in rumen fluid (P ≥ 0.11), but all were impacted by time (P < 0.01). A treatment × time interaction (P < 0.01) was detected for fecal pH and fecal pH change, with the pH being greater in TYL fed steers on d 56 and in CON fed steers on the final day. Steers that consumed CON had a reduced fecal pH (P < 0.01) and steers consuming SCFP tended to have a reduced fecal pH (P = 0.08) from d 35 to 56 compared with TYL fed steers, while both CON and SCFP fed steers experienced an increase in fecal pH from d 56 to 112 (P < 0.01). A time effect was detected (P ≤ 0.04) for plasma glucose, lactate, urea nitrogen, and non-esterified fatty acid concentrations among treatment groups. A treatment × time interaction was observed (P < 0.02) for stx2 genes of Shiga toxin producing Escherichia coli (STEC) in individual fecal samples where SCFP and TYL fed steers tended to exhibit an increased concentration compared with CON fed steers on d 14, while on d 190 CON and SCFP fed steers exhibited an increased concentration compared with TYL fed steers. Overall, STEC, E. coli O157:H7, and Salmonella concentrations were all impacted by time (P < 0.001). This experiment suggests that SCFP could be utilized as a potential alternative to TYL in feedlot cattle without adversely affecting cattle performance. However, SCFP did not impact pathogen shedding. In addition, CON fed steers performed similarly to both SCFP and TYL fed steers.

The effects of soil intake on the growth performance, rumen microbial community and tissue mineral deposition of German Mutton Merino sheep

Soil ingestion by livestock is common in grazing ecosystems, but few studies have been conducted to assess its effect on the animal organism. The topic is worthy of attention because these potential effects are likely to be enriched in the food chain and interfere with animal and human health. In this study, we present an indoor feeding trial conducted based on a completely randomized design to comprehensively evaluate the effects of simulated soil ingestion during grazing on nutrient digestibility, rumen fermentation, and microflora, and mineral deposition in the organs and tissues of sheep. Eighteen Mutton Merino crossbred sheep (42.7 ± 2.34 kg) were randomly allotted to three treatments and fed diets containing 0% (Control), 5% (SOIL5), and 10% (SOIL10) for 62 d, including a 7-d metabolism trial. It was found that soil intake altered the rumen fermentation in sheep, as evidenced by a decrease in total volatile fatty acids (VFA) and acetate concentrations in rumen fluid of 50.6% and 51.3%, respectively (p < 0.01), with soil proportion in the diet increased from 0% to 10%. Soil ingestion also reduced the species richness of rumen bacteria, with the relative abundance of Bacteroidetes decreasing significantly (p < 0.01), while that of Firmicutes and Proteobacteria increased considerably (p < 0.05). In terms of mineral elements deposition, higher levels of iron (Fe) were detected in the spleen and liver, and a higher concentration of copper (Cu) and zinc (Zn) in the liver were found in sheep fed a diet containing 5% soil compared to the other two groups (p < 0.05). Moreover, the concentrations of lead (Pb) in the liver and kidney, and arsenic (As) in the heart were also clearly increased after ingestion of soil (p < 0.05). Our findings indicate that although soil intake had no significant effect on the growth performance of sheep, it altered ruminal fermentation and increased the risk of excessive Fe, Pb, and As in their organism. This study supplies a theoretical basis for risk assessment of soil ingestion in grazing livestock.

Pengaruh Level Substitusi Rumput (Bothiriochola Pertusa) dengan Kangkung Terhadap pH, Konsentrasi VFA dan Amonia Cairan Rumen Ternak Kambing Kacang

This study was aimed to determine the effect of substitution level of Bothhriochloa pertusa grass with water spinach on pH, Volatile Fatty Acid, Concentration and ammonia in rumen fluid of peanut goat livestock. This study used 4 male kacang goats aged 8-1,5 years with a body weight range of 13,3-19,8 kg with an average of 16,2 kg. This study used a Latin Square design (RBSL) with four treatments and four replication periods. The four treatments were P0: Bothriochloa pertusa grass 60% + leucaena leucochephala 40%, P1: Bothriochloa pertusa grass 45% + leucaena leucochephala 40% + water spinach 15%, P2: Bothriochloa pertusa grass 30% + leucaena leucochephala 40% + water spinach 30%, P3: Botriochloa pertusa grass 15% + leucaena leucochephala 40% + water spinach 45%. Parameters measured were pH, Vollatile Fatty Acid (VFA) and Ammonia (NH3). The data obtained were analysed using Analisis of Variace (ANOVA. The results of this study indicate that pH, P0: 5.89 ± 0.22b, P1: 5,82 ± 0,1b, P2: 5,72 ± 0,17ab, P3: 5,57 ± 0,1a . VFA (mM), P0: 132,03 ± 3,99a, P1: 127,14 ± 3,99a, P2: 129,58 ± 6,31a, P3: 132,76 ± 4,95a. NH3 (mg/dl), P0: 15,89 ± 2,12a, P1: 15,14 ± 0,97a, P2: 15,52 ± 2,45a, P3: 16,41 ± 0,1a. The results of statistical analysis showed that the effect was not significant on the concentration of pH, concentration of VFA and rumen fluid ammonia of peanut goads. The conclusion of this study is that the substitusion of Bothriochloa pertusa grass with water spinach at different levels has the same effect between the treatments on pH, VFA concentration and rumen fluid ammonia of male peanut goat.

Effect of a garlic and citrus extract supplement on performance, rumen fermentation, methane production, and rumen microbiome of dairy cows

The aim of this trial was to determine the effect of a garlic and citrus extract supplement (GCE) on the performance, rumen fermentation, methane emissions, and rumen microbiome of dairy cows. Fourteen multiparous Nordic Red cows in mid-lactation from the research herd of Luke (Jokioinen, Finland) were allocated to 7 blocks in a complete randomized block design based on body weight, days in milk, dry matter intake (DMI), and milk yield. Animals within each block were randomly allocated to a diet with or without GCE. The experimental period for each block of cows (one for each of the control and GCE groups) consisted of 14 d of adaptation followed by 4 d of methane measurements inside the open circuit respiration chambers, with the first day being considered as acclimatization. Data were analyzed using the GLM procedure of SAS (SAS Institute Inc.). Methane production (g/d) and methane intensity (g/kg of energy-corrected milk) were lower by 10.3 and 11.7%, respectively, and methane yield (g/kg of DMI) tended to be lower by 9.7% in cows fed GCE compared with the control. Dry matter intake, milk production, and milk composition were similar between treatments. Rumen pH and total volatile fatty acid concentrations in rumen fluid were similar, whereas GCE tended to increase molar propionate concentration and decrease the molar ratio of acetate to propionate. Supplementation with GCE resulted in greater abundance of Succinivibrionaceae, which was associated with reduced methane. The relative abundance of the strict anaerobic Methanobrevibacter genus was reduced by GCE. The change in microbial community and rumen propionate proportion may explain the decrease in enteric methane emissions. In conclusion, feeding GCE to dairy cows for 18 d modified rumen fermentation and microbiota, leading to reduced methane production and intensity without compromising DMI or milk production in dairy cows. This could be an effective strategy for enteric methane mitigation of dairy cows.

A pilot investigation on the effect of induced saliva flow on digestive parameters in sheep, and a comparison with cattle.

Sheep with a relatively low methane yield were observed to have shorter fluid and particle mean retention times (MRT). Because the application of pilocarpine, a saliva stimulant, was successful in reducing retention times in ruminants in previous studies, we applied this substance to sheep, expecting a reduction in MRT and methane yield. Three non-pregnant sheep (74 ± 10 kg) were fed a hay-only diet in a 3 × 3 Latin square design with oral doses of 0, 2.5 and 5 mg pilocarpine/kg body weight and day. Measurements included feed and water intake, MRT of liquid and particulate phases in the reticulorumen (RR) and total gastrointestinal tract (GIT), ruminal microbial yield (via urinary purine bases and metabolic faecal nitrogen), total tract methane emission, apparent nutrient digestibility and rumen fluid parameters. Data were investigated for linear and quadratic effects using orthogonal polynomial contrasts. The MRT of liquid and small particles in the RR and total GIT, and the short-chain fatty acid concentration in rumen fluid, linearly declined with increasing pilocarpine dosage, while no quadratic relationship was detected. Intake of feed DM and water, apparent nutrient digestibility, methane yield and microbial yield were not affected by pilocarpine. When combining the sheep data with that of a similar experiment in cattle, we found that the MRT of the liquid phase was positively associated with estimated NDF digestibility and with methane production per digested NDF, but was not associated with microbial yield or the ratio of acetate to propionate. The ratio between MRT of the particulate and the liquid phase was smaller for sheep than that for cattle, and was not affected by treatment. Differences in this ratio might explain why species reacted differently to the saliva-inducing agent, which might help to explain the discrepancy between species in the effect of induced saliva flow on digestive parameters.

Effect of Supplementation of Nigella Sativa Oil on Nutrient Digestibility, Some Blood Metabolites and Rumen Parameters in Karadi Lambs

To examine the effect of supplementation of varying levels of the oil of Nigella sativa in Karadi lambs rations on nutrients digestibility, blood metabolites, and some rumen parameters, 18 Karadi lambs were allocated into three groups, and the first group was fed a basal diet as control whereas, the second (T2) and the third (T3) groups fed the basal diet being supplemented with 0.15 and 0.30% of DM Nigella sativa oil (NSO) respectively. All animals were fed individually on 1.5 kg/lamb/day. Results showed that dry matter (DM), crude protein (CP), organic matter (OM), and crude fiber (CF) digestibility was not affected (P&amp;gt;0.05) by NSO supplementation. Also, supplementing NSO had no significant effect on serum total protein (TP), albumin (Alb), globulin (Glb), triglycerides (TG), cholesterol (Chol), high density lipoprotein (HDL), and very low-density lipoprotein (VLDL) concentrations. There was an increasing trend (P=0.07) in LDL concentration of lambs fed on T2 and T3 as compared to control. Neither treatment nor interaction between time and treatment had an effect on rumen fluid pH. A significant decrease (P=0.008) was noted in rumen fluid pH value with the advances of time post feeding. The ammonia-nitrogen concentration in rumen fluid was generally lower upon oil supplementation, and it was significantly (P=0.03) decreased in the T2 group at 4 hours following morning feeding. It can be concluded that supplementing with 0.15 and 0.3% /DM of NSO showed a reduction in rumen ammonia-nitrogen while it had no effects on nutrient digestibility and blood metabolites in Karadi lambs.

Effects of oregano essential oil, cobalt and synergistic of both of them on rumen degradation rate and fermentation characteristics for corn silage

This study used oregano essential oil (EO), cobalt (Co) and synergistic of both of them additives instead of antibiotics to explore the effects of different additives on the corn silage fibre structure degradation, nutrient degradation rate and rumen fermentation characteristics of sheep. The additives used in the study as a product were provided by Ralco, Inc., USA. The Latin square experimental design was adopted with 5 treatments. The following treatments were fed through the rumen fistula: carrier (Car), carrier + EO, carrier + Co, carrier + EO + cobalt (EOC) and no additives as the control (CON). These treatments were fed to five sheep for five rounds at a dose of 80 mg/kg. At 48 h of degradation, the area of degraded corn leaf and husk reached 100%, SEM results showed that except for Car and CON treatment, there were obvious destroyed fibre structure differences under EO, Co and EOC treatment, showing total disappearance of leaf epidermal cells, palisade tissue and spongy tissue, especially under EOC. Under EOC treatment, DMD48 h, CPD48 h, NDFD48 h and ADFD48 h increased by 3.33%, 3.89%, 7.64% and 5.67%, respectively, compared with CON, while NDFD48 h and ADFD48 h increased by 4.70% and 6.05% under Co (p > 0.05). The NH3-N concentration in rumen fluid decreased by 19.36%, 27.20% and 19.48%, while the microbial protein (MCP) content increased by 6.32%, 4.29% and 5.97% (p > 0.05) under EO, Co and EOC treatments compared with CON at 48 h (p < 0.05). The total VFA concentration under EO, Co and EOC treatment was lower than CON at 6 and 48 h (p = 0.016, p = 0.022) and the propionate and acetate concentrations were higher under EOC treatment than CON at 48 h (p > 0.05). Feeding EOC increased the fibre structure degradation and nutrient degradation rate of corn silage and improved rumen fermentation in sheep, resulting in better effects than feeding EO or Co alone. HIGHLIGHTS This article revealed the significance of oregano essential oil synergistic with cobalt (EOC) on rumen fermentation characteristics and nutrient degradation rate of sheep. In this study, the degradation of leaves in the rumen was observed by scanning electron microscopy, and the degradation area was calculated by electron microscopy for the first time. The results of this study will provide the theoretical basis for EOC as a substitute for antibiotics.

Association between Ruminal pH and Rumen Fatty Acids Concentrations of Holstein Cows during the First Half of Lactation

Ruminal pH in dairy cows follows a diurnal fluctuation; low values are indicative of subacute ruminal acidosis and are associated with alterations of rumen fatty acids concentrations. The objective of the present study was to prospectively study the associations between ruminal pH and the rumen fluid concentrations of short, medium, and long chain fatty acids, under field conditions during the first half of lactation in 53 Holstein cows of a dairy farm. Ruminal fluid was obtained by rumenocentesis, which was performed at 30, 90, and 150 days in milk (DIM). Ruminal pH was measured immediately after collection with a portable pH meter, whereas gas chromatography was used for the determination of ruminal fatty acid concentrations. Mixed linear regression models were used for data analysis. The prevalence of cows with low ruminal pH (≤5.5) was 45.3%, 54.7%, and 66.0% at 30, 90, and 150 DIM, respectively. The concentrations of acetic, propionic, butyric, valeric, isovaleric, caproic and linoleic acids were negatively associated with ruminal pH values, whereas the acetic to propionic ratio was positively associated with rumen pH. Under field conditions and naturally occurring low ruminal pH cases, ruminal concentrations of most fatty acids are negatively related with ruminal pH values

Effects of hybrid Broussonetia papyrifera silage on growth performance, visceral organs, blood biochemical indices, antioxidant indices, and carcass traits in dairy goats

The crude protein content of hybrid Broussonetia papyrifera silage (HBPS) is close to that of alfalfa hay. In the present study, we substituted alfalfa hay with HBPS in diets for Saanen dairy goats, and tested a possibility of using HBPS as a feedstuff to provide protein. Thirty male Saanen dairy goats (6-months old, body weight 18.85 ± 1.44 kg) were divided into three groups of 10, and randomly assigned to three diets: the control group (CN) fed the basal diet containing 600 g/kg (dry matter (DM) basis) of alfalfa hay, and the other two groups fed the diets with 33 % and 67 % of alfalfa hay being substituted with HBPS, so HBPS accounted for 198 g/kg (LB) and 402 g/kg (HB) of the diet respectively. The experiment lasted for 94 days. Compared with CN, feeding HBPS did not change feed intake, body weight gain, and the visceral organ coefficients (P > 0.05), but reduced the feed conversion ratio and the weight gain cost, particularly on the 198 g/kg HBPS diet (P < 0.05). Serum concentrations of total protein, albumin, and urea-nitrogen in both LB and HB groups and glucose in HB group decreased (P < 0.05), but their values remained within the normal range. Ammonia concentration in rumen fluid was reduced by feeding HBPS (P < 0.05). The serum activity of superoxide dismutase (SOD) was increased and the concentration of malondialdehyde (MDA) decreased (P < 0.05) in both LB and HB groups (P < 0.05), and the SOD activity in longissimus dorsi muscle (LDM) was increased only for LB treatment (P < 0.05). Feeding HBPS had no effect on the carcass traits and water holding capacity in LDM (P > 0.05). In conclusion, HBPS had similar effects to alfalfa hay on the growth performances, visceral organ development, and water holding capacity postmortem, but reduced the feed conversion ratio and the feed cost, and enhanced the antioxidant capacity of animals. Inclusion of 198 g/kg of HBPS in the diet would be a better choice for growing goats. Reduced ammonia concentration in the rumen, urea and protein concentrations in blood suggest dietary protein digestion in the gastrointestinal tract could be affected by feeding HBPS, which is worthy of further investigation.

The effect of supplemented chestnut tannin to grass silage either at ensiling or at feeding on lamb performance, carcass characteristics and meat quality

This study was designed to investigate the effect of supplemented chestnut hydrolysable tannin (HT) both at ensiling and at feeding on lamb growth, carcass characteristic, and meat quality. Twenty tons of ryegrass (Lolium perenne) were used to produce silage. The ryegrass was treated at ensiling with one of three additives: 30 g kg-1 DM chestnut HT (GET), an inoculant as a positive control (GI), or water as a negative control (G). Another two treatments were made from ensiled grass by adding the 30 g kg-1 DM of chestnut HT to either positive (GI+T) or negative (GT) control. Forty Suffolk cross Mule lambs were used in this experiment and allocated to receive one of five experimental forage treatments with eight lambs per treatment. The diet consisted of two parts: concentrate and silage. Lambs were fed 215 g DM day-1 lamb of concentrate diet and ad libitum grass silage for seven weeks and then slaughtered. Back fat thickness tended to be lower (p= 0.07) for lambs fed the GT and GI+T treatments compared to lambs in the other experimental groups’ (10.0, 10.1, 9.8, 10.0, and 9.8 mm for GET, G, GT, GI, and GI+T, respectively). Feeding lambs GET tended to reduce (p= 0.06) meat lightness (L*) compared to the other treatments. Ammonia nitrogen concentration in rumen fluid was reduced significantly (p&lt; 0.05) when lambs consumed diets treated with tannin both at ensiling and at feeding (0.14, 0.19, 0.17, 0.17 and 0.14g l-1 GET G, GT, GI, and GI+T, respectively). The experimental treatments had no effect (p&gt; 0.05) on voluntary feed intake (914, 916, 899, 928, or 914 g day-1 for GET, G, GT, GI, and GI+T, respectively) or lamb performance.

Gluconeogenesis Alteration and p53-SIRT6-Fox01 Signaling Adaptive Regulation in Sheep from Different Grazing Periods.

The decline in sheep health and meat quality caused by seasonal nutritional deficiencies has always been an important problem in the production of naturally grazing sheep. Glucose metabolism is crucial in ruminants for adequate cell function and maintenance of the body tissues and systems. However, whether glucose metabolism, especially gluconeogenesis, is affected by seasonal grazing conditions has not been fully uncovered. Thus, twelve sheep from two seasons (dry and green grass periods) in natural grazing areas of Inner Mongolia, China, were selected for this study. Their serum glucose, insulin, PC, and PEPCK levels and volatile fatty acid (gluconeogenesis material) concentrations in rumen fluid were analyzed. The expression of key enzymes including PC, PEPCK, GLUT2, and G6P of gluconeogenesis and their regulators INSR, PI3K/AKT and p53-SIRT6-Fox01 in the liver was detected by real-time PCR and western blotting. The results revealed significant variances in gluconeogenesis and its indicators and showed p53-SIRT6-Fox01 as having potential regulation in different grazing periods. This study offers new insights into the mechanism of gluconeogenesis and adaptive regulation between dry grass period and green grass period and also provides a reference for maintaining the health of sheep and meat quality despite seasonal nutritional deficiencies.

Substituting ryegrass-based pasture with graded levels of forage rape in the diet of lambs decreases methane emissions and increases propionate, succinate, and primary alcohols in the rumen.

Feeding 100% forage rape to sheep consistently lowers methane emissions per unit of intake (CH4/DMI) compared to those fed 100% ryegrass pasture. However, forage rape is usually supplemented with other feeds, which might impact the mitigation potential provided by forage rape. The objective of this study was to determine the effect of substituting ryegrass with graded levels of forage rape in the diet of lambs on methane emissions and rumen fermentation characteristics. Seventy wether lambs (n = 14/treatment) were fed a ryegrass-based pasture substituted with 0%, 25%, 50%, 75%, and 100% of forage rape (Brassica napus; FR0, FR25, FR50, FR75, and FR100, respectively) on a dry matter basis. Methane emissions and dry matter intake were measured for 48 h in respiration chambers and a rumen fluid sample was collected. CH4/DMI decreased (P < 0.01) with increasing forage rape inclusion in the diet so that sheep fed FR100 and FR75 emitted 34% and 11% less, respectively, than those fed FR0. CH4/DMI differences for lambs fed FR25 and FR50 were much smaller (<6%) relative to FR0. The pH of rumen fluid decreased (P < 0.01) at higher levels of forage rape inclusion in the diet (FR75 and FR100) compared to low levels of inclusion (FR0, F25, and F50). The proportion of ruminal acetate was least in FR100 (30%) followed by FR75 (10%), FR50 (8%), and FR25 (4%) compared with FR0 (P < 0.001). The proportion of propionate plus succinate was greater for FR100 (+40%), FR75 (+28%), and FR50 (+29%) compared with FR0, with FR25 intermediate (P < 0.001). The methanol concentration, and ethanol and propanol proportions in rumen fluid were greater for FR100 compared with any other treatment (P < 0.001). In conclusion, CH4/DMI decreased at high levels of forage rape inclusion in the diet and especially feeding FR100 was associated with a pronounced shift in rumen fermentation profile, with a significant presence of succinate, ethanol, propanol, methanol, valerate, and caproate.

TECHNICAL NOTE: Analysis of volatile fatty acids in rumen fluid by gas chromatography mass spectrometry using a dimethyl carbonate extraction.

Analysis of rumen fluid volatile fatty acids (VFA) is typically conducted by injecting acidified aqueous rumen fluid into a gas chromatograph (GC) with a flame ionization detector (FID). Aqueous samples are highly problematic because of the large vapor volume that can lead to poor peak shape and contamination of inlets, potentially causing sample carryover. Methods using aqueous samples are not well suited for use in a mass spectrometer (MS) detector system. The objective of this project was to validate a dimethyl carbonate (DMC) extraction process and GCMS method for rumen VFA analysis. To perform the extraction, 100 µL of sample, KHSO4 (500 g/L), and 2-ethylbutyrate (internal standard; 8.5 mM) were added to a microcentrifuge tube (in order) followed by 1 mL of DMC. The mixture was thoroughly vortexed and centrifuged. The organic layer (top) was removed and placed in a GC vial. The DMC extract was injected (0.5 µL) into an Agilent 5977B GCMS (8:1 split injection) with a polar DB-FFAP column. The column was held at 105 °C for 5 min, increased at 10 °C/min to 150 °C, then 65 °C/min to 240 °C, and held constant for 10 min. The peak area of acetate relative to the internal standard is linear from approximately 2 mM to at least 130 mM and encompasses the expected values of rumen concentrations for the other VFA. Recovery of VFA from spiked rumen fluid was tested at three concentrations in rumen fluid from steers fed a finishing diet or grazing wheat pasture. Recovery was not affected by the diet of the animals (P > 0.10) or the amount of VFA spiked (P > 0.19) for acetate, propionate, isobutyrate, or butyrate. There was an interaction of amount of VFA spiked and the diet of the animal (P = 0.021) for valerate and a tendency for an interaction (P = 0.051) for isovalerate, due to the recovery of the VFA being lower in the medium spike amount in rumen fluid from cattle on wheat pasture. Overall, recovery was greatest for propionate (101.9 ± 1.67%) and lowest for valerate (95.7 ± 1.95%). Including the 10-min hold at 240 °C at the end of each run prevented carryover from sample to sample. This method appears to perform well in a GCMS system and accurately and precisely quantifies rumen fluid VFA.

Saccharomyces cerevisiae fermentation products reduce bacterial endotoxin concentrations and inflammation during grain-based subacute ruminal acidosis in lactating dairy cows

Subacute ruminal acidosis (SARA) is a metabolic disorder in dairy cows that is associated with dysbiosis of rumen and hindgut microbiomes, translocation of immunogenic compounds from the gut lumen into blood circulation, and systemic inflammatory response. In this study we hypothesized that Saccharomyces cerevisiae fermentation products (SCFP) attenuate the increases in ruminal and peripheral bacterial endotoxin concentrations and the inflammation resulting from repeated induction of SARA. Lactating Holstein dairy cows (parity 2 and 3+, n = 32) were fed diets with or without SCFP (all from Diamond V) and subjected to 2 episodes of SARA challenges. Cows received a basal total mixed ration (TMR) containing 34% neutral detergent fiber and 18.6% starch, dry matter (DM) basis. Treatments were randomly assigned to control (basal TMR and 140 g/d of ground corn with no SCFP) or 1 of 3 SCFP treatments: basal TMR and 14 g/d Original XPC (SCFPa), 19 g/d NutriTek (SCFPb-1×), or 38 g/d NutriTek (SCFPb-2×) mixed with 126, 121, or 102 g/d of ground corn, respectively. Treatments were implemented from 4 wk before until 12 wk after parturition. During wk 5 (SARA1) and wk 8 of lactation (SARA2), grain-based SARA challenges were conducted by gradually replacing 20% of DM of the basal TMR over 3 d with pellets containing 50% wheat and 50% barley. Ruminal fluid, fecal, and blood samples were collected weekly during Pre-SARA1 (wk 4, as baseline), Post-SARA1 (wk 7), and Post-SARA2 (wk 10 for blood and wk 12 for rumen and fecal parameters) stages, and twice a week during the challenges SARA1 and SARA2. Rumen papillae samples were taken only during Pre-SARA1 and Post-SARA2. We measured the concentrations of free lipopolysaccharides (LPS) in the rumen fluid and feces; free LPS and lipoteichoic acid (LTA) endotoxins in peripheral plasma; interleukin (IL)-1β and IL-6 in peripheral serum; acute-phase proteins, serum amyloid A (SAA), and LPS-binding protein in peripheral plasma; haptoglobin (Hp) in peripheral serum; and myeloperoxidase (MPO) in rumen papillae. Induction of SARA episodes increased free LPS concentrations in rumen fluid and tended to increase LTA in peripheral plasma. The SARA episodes increased concentration of circulating SAA and tended to increase that of IL-1β compared with Pre-SARA1. Induction of SARA did not affect the concentrations of circulating IL-6, Hp, and MPO. The SCFP supplementation reduced plasma concentrations of LTA and SAA and serum concentration of IL-1β compared with control. Additionally, SCFPb-2× tended to reduce ruminal LPS in second-parity cows compared with control. Overall, SCFP supplementation appeared to stabilize the rumen environment and reduce proinflammatory status, hence attenuating adverse digestive and inflammatory responses associated with SARA episodes.

The impact of Fusarium mycotoxins on macro and micro parameters of dairy cattle

The aim of the present study was to evaluate the impact on macro and micro parameters of dairy cattle when cows consumed total mixed ration (TMR) contaminated with (zearalenone) ZEA and deoxynivalenol (DON) for 60 days. Macro indicators included milk composition and production and micro parameters included blood components, rumen volatile fatty acids (VFAs), and ZEA and DON concentrations in rumen fluid and milk. Twenty dairy cows were fed TMR with naturally contaminated mycotoxins (ZEA 300 - 500 μg/kg; DON 102 - 200 μg/kg). The trial lasted for 60 days. Samples of blood, rumen fluid, and milk were taken three times at intervals of 30 days. The obtained results showed that the combination of ZEA and DON affected liver and kidney enzymes (p < 0.05) because, in 60 days, alkaline phosphatase (ALP) increased by 25%, albumin (ALB) decreased by 8.7%, creatinine (CREA) decreased by 40%. Rumen ammoniacal nitrogen (NH$_{3}$-N) statistically significantly increased (43%), whereas acetate acid decreased (2.7%). In 60 days, the combination of mycotoxins ZEA and DON statistically significantly reduced the milk yield (20.8%), fats (11.8 %), and proteins (3.5%). After 60 days ZEA in rumen fluid increased (28.6%), DON increased (30%) (p > 0.05). In milk samples, ZEA concentration increased from 0.97 μg/L (0th day) to 1.43 μg/L (on the 60th day). Based on the obtained results, it was determined that combination of Fusarium mycotoxins occurring in the fodder produced under Lithuanian climatic conditions significantly affected some macro and micro indicators.

Comparison of HPLC and NMR for quantification of the main volatile fatty acids in rumen digesta

Accurate quantification of volatile fatty acid (VFA) concentrations in rumen fluid are essential for research on rumen metabolism. The study comprehensively investigated the pros and cons of High-performance liquid chromatography (HPLC) and 1H Nuclear magnetic resonance (1H-NMR) analysis methods for rumen VFAs quantification. We also investigated the performance of several commonly used data pre-treatments for the two sets of data using correlation analysis, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). The molar proportion and reliability analysis demonstrated that the two approaches produce highly consistent VFA concentrations. In the pre-processing of NMR spectra, line broadening and shim correction may reduce estimated concentrations of metabolites. We observed differences in results using multiplet of different protons from one compound and identified “handle signals” that provided the most consistent concentrations. Different data pre-treatment strategies tested with both HPLC and NMR significantly affected the results of downstream data analysis. “Normalized by sum” pre-treatment can eliminate a large number of positive correlations between NMR-based VFA. A “Combine” strategy should be the first choice when calculating the correlation between metabolites or between samples. The PCA and PLS-DA suggest that except for “Normalize by sum”, pre-treatments should be used with caution.

PSVII-17 Use of a dimethyl carbonate extraction for rumen fluid volatile fatty acid quantification by gas chromatograph mass spectrometry

Abstract Analysis of rumen fluid volatile fatty acids (VFA) is typically conducted by injecting acidified aqueous rumen fluid into a gas chromatograph (GC) with a flame ignition detector (FID). Water in GC samples can lead to poor peak shape and to contamination of inlets, potentially causing sample carryover. Aqueous methods are not well suited for use in mass spectrometer (MS) detector systems. The objective of this project was to validate a dimethyl carbonate (DMC) extraction process and GCMS method for rumen VFA analysis. To perform the extraction, 100 µL of sample, KHSO4 (50 g/L), and 2-ethylbutyrate (internal standard; 0.86 mM) are added to a microcentrifuge tube (in order) followed by 1 mL of DMC. The mixture is thoroughly vortexed and centrifuged. The organic layer (top) is removed and placed in a GC vial. The DMC extract is injected (0.5 µL) into an Agilent 5977B GCMS (8:1 split injection) with a polar DB-FFAP column. The column was held at 105°C for 5 min, increased at 10°C/min to 150°C, then 65°C/min to 240°C, and held constant for 10 min. The assay is linear for acetate from approximately 2 mM to at least 130 mM and covers the expected values of rumen concentrations for the other VFA. Recovery of VFA from spiked rumen fluid was tested at three concentrations in rumen fluid from steers fed a finishing diet or grazing wheat pasture. Recovery was not affected by the diet of the animals (P &amp;gt; 0.19) or the amount of VFA spiked (P &amp;gt; 0.27) and averaged 99.9% for all VFA, with valerate being the lowest (95.9%). Including the 10 min hold at 240°C at the end of each run prevented carryover from sample to sample. This method appears to perform well in a GCMS system and accurately and precisely quantify rumen fluid VFA.