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Digestibility trials need a viable rumen fluid as inoculum to degrade feeds. The variability of rumen fluid depends on the animal’s diet, while its viability is greatly influenced by the sampling and handling procedures. In this article, we present a replicable protocol for sampling the rumen fluid from slaughtered animals for in vitro digestibility trials. A detailed list of the tools and a step-by-step standardized procedure for the collection, storage and the transportation of the rumen fluid from the slaughterhouse to the laboratory is presented. We also describe a digestibility trial for establishing the maximum storage time of rumen fluid from sampling to its use. The results show that the rumen fluid, collected and maintained according to the proposed protocol, can be stored and used from 30 to 300 min from sampling without significantly compromising the fermentative activity of the microbial population.

Abstract Background Rumen juice analysis (RJA) involves analysis of a 10mL sample within minutes after sampling. However, it can be challenging to collect 10 mL of rumen juice (RJ) from some ruminants, and clinical circumstances can delay RJA. Objectives Quantify the effect of sample volume (2, 5, 10, 50, and 100 mL), and time-to-analysis (0, 30, and 60 minutes) on RJA. Animals Cannulated cow. Methods Observational experimental study. Two liters of RJ were collected at 26 separate times. The samples were subdivided into 2 duplicates of each sample volume at each sampling time; and analyzed at 0, 30, and 60 minutes after collection. Rumen juice analysis included pH measurement, methylene blue reduction time (MBRT), and protozoal motility. Results The pH of 2 and 5 mL samples was significantly (P = .01) higher than the pH of 50 and 100 mL samples at all time points. The MBRT was significantly lower (faster bacterial reduction) for 100 mL samples compared to all other samples at 0 minute and to 2, 5, and 50 mL samples at 30 min. The pH and MBRT at 60 minutes were significantly higher than at 0 minute for all volumes (P < .05 and P < .01, respectively). For large protozoa, small sample volumes (2 and 5 mL) had significantly lower protozoal motility (scores of 5 and 4.5, respectively) compared to 100 mL samples at 60 minutes (score of 4; P < .05). Conclusions and Clinical Importance Interpretation of RJA could be affected by small sample volumes and delays to analysis. Sample volumes of ≥10 mL analyzed within 30 minutes after collection are recommended.

The objective of this study was to systematically investigate how sodium butyrate (SB) affects the gastrointestinal bacteria in newborn calves at different stages before weaning. Forty female newborn Holstein calves (4-day-old, 40 ± 5 kg of body weight) were randomly divided into four groups; each group was supplemented with four SB doses: 0, 15, 30, and 45 g/day (ten replicates) in SB0, SB15, SB30, and SB45 groups, respectively. SB was fed with milk replacer from day 4 to day 60. Rumen fluid and feces were collected on days 2, 14, 28, 42, and 60 for 16S rRNA high-throughput sequencing. Data were analyzed in a complete randomized design and analyzed on the online platform of Majorbio Cloud Platform. The results showed that SB significantly increased the α-diversity in feces, especially Shannon and Chao indices in SB45 and SB30 at day 60 more than in SB15 (P < 0.05). Additionally, SB significantly enhanced Firmicutes growth from day 2 to 28 and also increased Bacteroides abundance from day 28 to 42 in rumen and feces (P < 0.05). SB also significantly inhibited Proteobacteria abundance in rumen and feces during the study period (P < 0.05). SB also promoted some potential beneficial bacterial abundance, including Prevotella, Lachnospiraceae, Clostridium, Ruminococcus, and Muribaculaceae (P < 0.05). Additionally, Escherichia-Shigella abundance at SB0 was significantly lower than in the other groups (P < 0.05). In conclusion, this study firstly reported a dynamic curve showing of the SB effects on bacteria in calves before weaning. This study provides valuable evidence for the development of the gastrointestinal tract of the calves in the early stage of the life. SB supplementation improved the gastrointestinal health by regulating the bacterial populations.Key points• The gastrointestinal tract of calves has been improved after the SB supplementation.• Microbes were the vital influential factor in the development of calves.• Intervention before weaning is an effective strategy for calf health.

Fermentative kitchen waste to produce high-value chemicals (e.g., acetic acid) has been investigated actively in the past decades. Creating an alkaline condition is widely used to improve the hydrolysis of polysaccharide and inhibit the methanogenesis, but this method significantly increases the overall cost. Herein, the present study investigated the bioaugmentation with rumen fluid to improve acetic acid production from kitchen waste at neutral condition via strengthening hydrolytic and acid-forming bacteria. Results showed that the highest acetic acid yield reached 1.52 g/L at rumen fluid and granular sludge ratio of 1:1. The proportion of acetic acid in volatile fatty acids (VFAs) has increased by 10% compared to control. Microbial community analysis revealed that bioaugmentation with rumen fluid increased the relative abundance of Prevotella and Rikenellaceae_RC9_gut_group which has the ability to degrade polysaccharides and produce acetic acid. Moreover, the proliferation of butyric acid producers (Clostridium_sensu_stricto_1 and Clostridium_sensu_stricto_7) were inhibited significantly, which was in agreement with high acetic acid proportion in VFAs. The bioaugmentation strategy and process optimization provided an energy and cost-saving method for acetic acid production from kitchen waste.

Rumen cud transfaunation re-establishes rumen micro environment and improves fermentation in recipient animals affected with digestive disorders. Preserving rumen cud or fluid will increase its availability for the treatment of rumen fermentation disorders, without having to maintain donor animals. Rumen fluid collected from healthy goats, fed standard ration having roughage 70% and concentrate 30%, was lyophilized (prefreezing -80 °C, 48 h; lyophilization -45 °C, 32 h) using 5% glycerol as cryoprotectant. The 16 S metagenome analysis of the lyophilized rumen fluid (LRF) revealed an abundance of Prevotella (33.2%). Selenomonas ruminantium (1.87%) and Megasphaera elsdenii (0.23%) were also present. Twenty-four goats having history of high grain feeding and exhibiting clinical symptoms of rumen fermentation disorders were randomly distributed into either one of the two treatment groups viz . , T 1  = oral administration of LRF 31 g/animal/day and T 2  = oral administration of sodium bicarbonate (SB) 15 g/animal/day. Post intervention LRF and SB, improved animal body condition, feed intake, fecal consistency, elevated the ruminal pH at 48 h, reduced propionate and lactate at 48 h, reduced total volatile fatty acids (TVFA) and ammonia nitrogen at 24 h. Significant reduction in serum blood urea nitrogen (BUN) and urea levels were observed even from 24 h post intervention irrespective of the treatments. LRF significantly improved acetate and decreased propionate production compared to SB. LRF at 7.5% (v/v) can thus be used to counteract ruminal fermentation disorders in goats sequel to high grain ration.

Objective: The aim of this study was to examine the solvent conditions suitable for preparing samples of total mixed ration (TMR) silage and ruminal fluid, with or without chitosan inclusion, to achieve more sensitive, specific, and repeatable analyses. Material and Methods: The metabolome contained in the sample was determined using an ultra-high-performance liquid chromatography-high-resolution mass spectrometry quadrupole using samples in the form of TMR silage (silage with 0.5% inclusion of chitosan, silage without chitosan addition) and rumen fluid (rumen fluid containing 0.5% chitosan, ruminal fluid without chitosan) with and without the addition of chitosan, as well as three different solvent ratios: 50% water MS grade: 50% methanol (MeOH) MS grade (1), 20% water MS grade: 80% MeOH MS grade (2), and 0% water MS grade: 100% MeOH MS grade (3). Results: The TMR silage samples contained 311 metabolites, amino acid compounds predominating, followed by fatty acids and amines. Fatty acids, organic phosphate, and amines dominated the 39 metabolites found in rumen fluid samples. In the TMR silages, 100% MeOH seemed to be able to distinguish samples more effectively than other solvents; moreover, cinnamic acid, D-(+)-proline, and L(+)-ornithine were the three highest variable importance for projection values among prospective metabolite markers for silage samples. Whereas the use of a 50% MeOH to 50% water solvent ratio achieved the best discrimination results in rumen fluid samples, the three highest variable importance for projection values among prospective metabolite markers for ruminal fluid samples were triethyl phosphate, dibenzylamine, and phosphoric acid. Conclusion: 100% MeOH is able to distinguish TMR silage, while 50% MeOH to 50% water is best for ruminal fluid samples.

To identify differences in rumen function as a result of feeding monensin to beef cattle, rumen fluid metagenomics and metabolomics analyses were used to evaluate the functional attributes and metabolites of rumen microbiota in beef steers fed no or 200 mg/d of monensin. Eight rumen-fistulated steers were used in the study for a period of 53 days. Rumen fluid samples were collected on the last day of the experiment. Monensin increased the relative abundance of Selenomonas sp. ND2010, Prevotella dentalis, Hallella seregens, Parabacteroides distasonis, Propionispira raffinosivorans, and Prevotella brevis, but reduced the relative abundance of Robinsoniella sp. KNHs210, Butyrivibrio proteoclasticus, Clostridium botulinum, Clostridium symbiosum, Burkholderia sp. LMG29324, and Clostridium butyricum. Monensin increased the relative abundance of functional genes involved in amino acid metabolism and lipid metabolism. A total of 245 metabolites were identified. Thirty-one metabolites were found to be differentially expressed. Pathway analysis of the differentially expressed metabolites revealed upregulated metabolic pathways associated with metabolism of linoleic acid and some amino acids. These findings confirm that monensin affects rumen fermentation of forage-fed beef cattle by modulating the rumen microbiome, and by reducing amino acid degradation and biohydrogenation of linoleic acid in the rumen.

Alpaca ( Vicugna pacos ) is a South American camelid that was introduced recently to Egypt and considered a zoo animal in different country zoos. There are no available data regarding the normal values of rumen fluid and hematobiochemical constituents of alpacas bred in Egypt. The aim of this study was to estimate normal values for rumen fluid and blood constituents of alpaca and determine the influence of season and sex on these parameters under Egyptian circumstances. The study was conducted on seventeen (8 female and 9 male); 2–6 years apparently healthy alpacas. Rumen fluid and blood samples were taken from each animal (from August 2022 to February 2023) and divided into summer and winter samples. Rumen fluid constituents were influenced by seasonal changes and showed a significant ( p  < 0.05) increase in the total protozoal count in summer, while in winter, rumen fluid pH and ammonia values were significantly ( p  < 0.05) increased. For hematology, the effect of season was evident in the red blood cells count and packed cell volume, which increased significantly ( p  < 0.05) in winter. Regarding serum biochemistry, winter showed significant ( p  < 0.05) elevation in glucose, urea and magnesium levels, while summer had significantly ( p  < 0.05) increased chloride levels. The effect of sex was minimal, and only glucose and creatinine values showed significant ( p  < 0.05) increases in males compared to females. The effect of season was evident in rumen fluid, hematology and serum biochemical parameters of alpaca, while sex has minimal effect on these parameters. Rumen fluid and hematobiochemical constituent values of apparently healthy alpacas bred under Egyptian conditions were consistent with those in other countries, while slight differences were observed in total protozoal count and serum minerals. The data obtained in this study can be used as preliminary data for the rumen fluid and hematobiochemical constituents of alpacas kept under Egyptian circumstances.

This study was aimed to investigate the effects of adding different concentrations of yeast on some of rumen fluid traits and some blood characteristics in adult Awassi lambs. The study was conducted in the animal house at the College of Veterinary Medicine/University of Tikrit for the period from 15/7/2017 to 1/10/2017, using 16 Awassi lambs, aged (10 – 12 months), and an average weight of (36 kg)., The animals were divided into four groups, each one included four lambs. The first group was considered as control (T1), without the addition of bread yeast to their foods. The diets in the other groups, (T2), (T3) and (T4) contained the  yeast in the proportions of 3, 5 and 7 gm/ animal / days, respectively. Wheat straw was introduced as a free coarse feed as well as concentrated feed at 2.5% of weight, which is measured weekly for 75 days. The study showed no significant differences in the concentration of ammonia, volatile fatty acids and pH, measured after 1 and 3 hours with feed. While the pH was significant (P≤0.05) immediately after feeding, no significant differences were found in all studied blood characteristics except, which are the levels of cholesterol and total protein ,which was differ significantly (P≤0.05) .

Monitoring rumination activity is considered a useful indicator for the early detection of diseases and metabolic disorders. Accelerometer-based sensor systems provide health alerts based on individual thresholds of rumination times in dairy cows. Detailed knowledge of the relationship between sensor-based rumination times and rumen physiology would help detect conspicuous animals and evaluate the treatment’s success. This study aimed to investigate the association between sensor-based health alerts and rumen fluid characteristics in Holstein-Friesian cows at different stages of lactation. Rumen fluid was collected via a stomach tube from 63 pairs of cows with and without health alerts (ALRT vs NALRT). Pairs were matched based on the day of lactation, the number of lactations, and health criteria. Rumen fluid was collected during and after health alerts. The parameters of color, odor, consistency, pH, redox potential, sedimentation flotation time, and the number of protozoa were examined. Results showed differences between both groups in odor, rumen pH, sedimentation flotation time, and protozoan count at the first rumen fluid collection. Within the groups, greater variations in rumen fluid parameters were found for ALRT cows compared to NALRT cows. The interaction between health alert and stage of lactation did not affect the rumen fluid parameters.