The effects of residual energy intake on nutrient use, methane emissions and microbial composition in dairy cows.

For sustainable food production selection and breeding of feed efficient animals is crucial. The objective of this study was to evaluate whether multiparous dairy cows, ranked during their first lactation based on residual energy intake (REI) as efficient (low; L-REI) or inefficient (high; H-REI), differ in terms of nutrient use efficiency, methane emissions, rumen fermentation, and gut microbiota composition. Six L-REI and 6 H-REI cows were offered two diets with either a low or high proportion of concentrates (30 vs. 50% of DM) on two consecutive periods of 21 d. Gas exchanges, milk yield, feces and urine excretions were measured in open-circuit respiratory chambers. The results indicated that L-REI cows had higher methane yields (22.6 vs. 20.4 g/kg DM intake) and derived more energy (energy balance - 36.6 vs. - 16.9 MJ/d) and protein (N balance - 6.6 vs. 18.8 g/d) from the tissues to support similar milk yields compared to H-REI cows. Nutrient intake and digestibility were not affected by REI, and there were no interactions between REI and diet. Milk yield, milk production efficiency, and milk composition were not affected by REI except for milk urea concentration that was higher for L-REI cows (14.1 vs. 10.8 mg/100 ml). The rumen and fecal microbiota community structure and function were associated with both the diet and REI, but the diet effect was more pronounced. The current study identified several physiological mechanisms underlying the differences between high and low REI cows, but further studies are needed to distinguish the quantitative role of each mechanism.

Methane prediction equations including genera of rumen bacteria as predictor variables improve prediction accuracy.

Methane (CH4) emissions from ruminants are of a significant environmental concern, necessitating accurate prediction for emission inventories. Existing models rely solely on dietary and host animal-related data, ignoring the predicting power of rumen microbiota, the source of CH4. To address this limitation, we developed novel CH4 prediction models incorporating rumen microbes as predictors, alongside animal- and feed-related predictors using four statistical/machine learning (ML) methods. These include random forest combined with boosting (RF-B), least absolute shrinkage and selection operator (LASSO), generalized linear mixed model with LASSO (glmmLasso), and smoothly clipped absolute deviation (SCAD) implemented on linear mixed models. With a sheep dataset (218 observations) of both animal data and rumen microbiota data (relative sequence abundance of 330 genera of rumen bacteria, archaea, protozoa, and fungi), we developed linear mixed models to predict CH4 production (g CH4/animal·d, ANIM-B models) and CH4 yield (g CH4/kg of dry matter intake, DMI-B models). We also developed models solely based on animal-related data. Prediction performance was evaluated 200 times with random data splits, while fitting performance was assessed without data splitting. The inclusion of microbial predictors improved the models, as indicated by decreased root mean square prediction error (RMSPE) and mean absolute error (MAE), and increased Lin's concordance correlation coefficient (CCC). Both glmmLasso and SCAD reduced the Akaike information criterion (AIC) and Bayesian information criterion (BIC) for both the ANIM-B and the DMI-B models, while the other two ML methods had mixed outcomes. By balancing prediction performance and fitting performance, we obtained one ANIM-B model (containing 10 genera of bacteria and 3 animal data) fitted using glmmLasso and one DMI-B model (5 genera of bacteria and 1 animal datum) fitted using SCAD. This study highlights the importance of incorporating rumen microbiota data in CH4 prediction models to enhance accuracy and robustness. Additionally, ML methods facilitate the selection of microbial predictors from high-dimensional metataxonomic data of the rumen microbiota without overfitting. Moreover, the identified microbial predictors can serve as biomarkers of CH4 emissions from sheep, providing valuable insights for future research and mitigation strategies.

Risky Sexual Behaviors and Condom Use Barriers in Iranian Women with Substance Use Disorders.

Background: Scant information exists on Iranian women's protective behaviors mainly constant condom use. Inconsistent condom use seems prevalent among women with substance use problems. We aimed to investigate risky sexual behaviors (RSBs) and condom use barriers in Iranian women with substance use disorders (SUDs). Methods: In our cross-sectional study, we recruited 300 women who sought treatments for their SUDs from the active outpatient drug free (ODF) and Methadone Maintenance Therapy (MMT) centers in Tehran, Iran during 2017-2021. We used three batteries including demographic questionnaire, the Risky Sexual Behavior Questionnaire (RSBQ); and the Condom Barriers Scale (CBS). The statistical software R, analysis of variance post hoc and multivariate analysis of variance (MANOVA) logistic regression tests were used in data analysis. Results: The majority reported at least one lifetime experience of RSBs. Our results show that only 22% of the participants 'always' use condom in their sexual encounters. The lowest and highest subscale scores of the CBS were related to Sexual Experience (SE) (2.47 ± 0.86) and access/availability structure (3.52 ± 0.7), respectively. RSBs had negative significant association with Partner Barrier (PB) subscale scores (OR = 0.4; 95% CI: 0.22 to 0.73) and effect on SE subscale scores (OR= 0.54; 95% CI: 0.31 to 0.94). Conclusion: RSBs was prevalent among our study population. RSBs and condom use barriers are significantly interwoven. The condom use barriers were highly associated with the types of sexual encounters such as group sex or casual sexual relations than specific mean of sexual performance (i.e. anal sex). Gender-specific RSBs, STIs/HIV/AIDS prevention program is recommended for women with SUDs.

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.

Integrating heterogeneous across-country data for proxy-based random forest prediction of enteric methane in dairy cattle.

Direct measurements of methane (CH4) from individual animals are difficult and expensive. Predictions based on proxies for CH4 are a viable alternative. Most prediction models are based on multiple linear regressions (MLR) and predictor variables that are not routinely available in commercial farms, such as dry matter intake (DMI) and diet composition. The use of machine learning (ML) algorithms to predict CH4 emissions from across-country heterogeneous data sets has not been reported. The objectives were to compare performances of ML ensemble algorithm random forest (RF) and MLR models in predicting CH4 emissions from proxies in dairy cows, and assess effects of imputing missing data points on prediction accuracy. Data on CH4 emissions and proxies for CH4 from 20 herds were provided by 10 countries. The integrated data set contained 43,519 records from 3,483 cows, with 18.7% missing data points imputed using k-nearest neighbor imputation. Three data sets were created, 3k (no missing records), 21k (missing DMI imputed from milk, fat, protein, body weight), and 41k (missing DMI, milk fat, and protein records imputed). These data sets were used to test scenarios (with or without DMI, imputed vs. nonimputed DMI, milk fat, and protein), and prediction models (RF vs. MLR). Model predictive ability was evaluated within and between herds through 10-fold cross-validation. Prediction accuracy was measured as correlation between observed and predicted CH4, root mean squared error (RMSE) and mean normalized discounted cumulative gain (NDCG). Inclusion of DMI in the model improved within and between-herd prediction accuracy to 0.77 (RMSE = 23.3%) and 0.58 (RMSE = 31.9%) in RF and to 0.50 (RMSE = 0.327) and 0.13 (RMSE = 42.71) in MLR, respectively than when DMI was not included in the predictive model. When missing DMI records were imputed, within and between-herd accuracy increased to 0.84 (RMSE = 18.5%) and 0.63 (RMSE = 29.9%), respectively. In all scenarios, RF models out-performed MLR models. Results suggest routinely measured variables from dairy farms can be used in developing globally robust prediction models for CH4 if coupled with state-of-the-art techniques for imputation and advanced ML algorithms for predictive modeling.

Measuring Sexual Performance: Development and Psychometric Properties of the Sexual Performance Questionnaire in Iranian People with Spinal Cord Injury.

Sexual performance refers to activities served by sexual capacity and motivation. As a culturally sensitive issue, to date the scalar invariance of sexual performance has not been examined for Iranians with spinal cord injuries (SCIs). Aim: To develop and assess properties of an instrument evaluating sexual performance of an Iranian population with SCIs., in Brain and Spinal Cord Injury Research Center (BASIR), Neuroscience Institute, Tehran University of Medical Sciences, Tehran, Iran. Using multi-modal methods, we developed and assessed the Sexual Performance Questionnaire (SPQ). This included collecting expert opinions; engaging with patients with SCI referred to BASIR; pilot testing to assess the scale; and a formal investigation. Participants (men = 156, women = 58) completed the SPQ. Internal consistency and reliability were measured using Cronbach's α coefficient. Content and face validity were examined by academic experts. Construct validity was assessed by examining convergent and discriminant validity. Finally, exploratory factor analysis was used to extract the factor structure of the questionnaire. The Cronbach's α coefficient was 0.77. There was a significant (p = 0.04) correlation (r = - 0.23) between the SPQ score and age. Those with a partner scored higher (p = 0.001). We found three components: Spouse as initiator, self-initiation, and genital-oriented sex which accounted for 59% of the observed variance. The face and content validity was approved by an expert committee. The development and application of the 13-item SPQ provides a thorough understanding of sexual performance amongst persons with SCI. It facilitates the development of efficient sexual rehabilitation interventions and SCI-specific sexuality education programs.

Effects of Starch Level and a Mixture of Sunflower and Fish Oils on Nutrient Intake and Digestibility, Rumen Fermentation, and Ruminal Methane Emissions in Dairy Cows.

Four multiparous dairy cows were used in a 4 × 4 Latin square to examine how starch level and oil mixture impact dry matter (DM) intake and digestibility, milk yield and composition, rumen fermentation, ruminal methane (CH4) emissions, and microbial diversity. Experimental treatments comprised high (HS) or low (LS) levels of starch containing 0 or 30 g of a mixture of sunflower and fish oils (2:1 w/w) per kg diet DM (LSO and HSO, respectively). Intake of DM did not differ between cows fed LS and HS diets while oil supplementation reduced DM intake. Dietary treatments did not affect milk and energy corrected milk yields. There was a tendency to have a lower milk fat concentration due to HSO compared with other treatments. Both high starch level and oil supplementation increased digestibility of gross energy. Cows receiving HS diets had higher levels of total rumen VFA while acetate was lower than LS without any differences in rumen pH, or ruminal CH4 emissions. Although dietary oil supplementation had no impact on rumen fermentation, decreased CH4 emissions (g/day and g/kg milk) were observed with a concomitant increase in Anoplodinium-Diplodinium sp. and Epidinium sp. but a decrease in Christensenellaceae, Ruminococcus sp., Methanobrevibacter ruminantium and Mbb. gottschalkii clades.

Residual carbon dioxide as an index of feed efficiency in lactating dairy cows.

High feed costs make feed conversion efficiency a desirable target for genetic improvement. Residual feed intake (RFI), calculated as the difference between observed and predicted intake, is a commonly used estimate of feed efficiency. However, determination of feed efficiency in dairy herds is challenging due to difficulties in measuring feed intake of individual animals reliably. Using residual CO2 (RCO2) production as an estimate of feed efficiency would allow ranking the cows according to feed efficiency, provided that CO2 production is closely related to heat production and feed intake. The objective of this study was to evaluate the potential of RCO2 as an index of feed efficiency using data from respiration calorimetry studies (289 cow per period observations). Heat production was precisely predicted from CO2 production [root mean square error (RMSE)] adjusted for random effects was 1.5% of observed mean]. Dry matter intake (DMI) was better predicted from energy-corrected milk (ECM) yield and CO2 production than from ECM yield and body weight in the model (adjusted RSME = 0.92 vs. 1.39 kg/d). Residual CO2 production estimated as the difference between actual CO2 production and that predicted from ECM yield, metabolic body weight was closely related to RFI (adjusted RMSE = 0.42) that was calculated as the difference between actual DMI and that predicted from ECM, metabolic body weight, and energy balance (EB). When the cows were categorized in 3 groups of equal sizes on the basis of RCO2 (low, medium, and high), low RCO2 cows had lower DMI, RFI, methane production and intensity (g/kg ECM), and heat production, but higher efficiency of metabolizable energy utilization for lactation than high RCO2 cows. When RFI was predicted from RCO2, the residuals (observed - predicted) were negatively related to EB and digestibility. Predicting RFI with a 2-variable model based on RCO2 and digestibility, adjusted RMSE decreased to 0.23 kg/d, and residuals were not significantly related to EB. The cows in low RCO2 group had a higher energy digestibility than the cows in the high RCO2 group, and differences in EB were observed between the groups. Error of the model predicting residual ECM production from RCO2 was 1.41 kg/d. The residuals were positively related to ECM yield and energy digestibility. Predicting residual ECM from RCO2 and ECM yield decreased adjusted RMSE to 1.07 kg/d, and further to 0.78 kg/d when digestibility was included in the 2-variable model. It is concluded that RCO2 has a potential for ranking individual cows based on feed efficiency.

Comparing thoracoabdominal injuries of manual versus load-distributing band cardiopulmonary resuscitation.

OBJECTIVE: To identify and compare manual and load-distributing band (LDB) cardiopulmonary resuscitation (CPR)-related injuries. METHODS: Retrospective observational cohort study. Adult, nontraumatic deaths with a postmortem computed tomography scan (PMCT) performed were classified into two groups: deceased after LDB CPR or after manual CPR. PMCT scans were reviewed for thoracoabdominal injuries such as fractures, pneumothorax and hemorrhage. The injuries between groups were compared. RESULTS: LDB CPR (n = 43) showed increased incidences of posterior rib fractures (53 vs 18%, P = 0.006), pneumothorax (23 vs 4%, P = 0.04) and more pericardial fluid (median 12 vs 6 mm, P = 0.002) compared with manual CPR (n = 29). Multivariable regression analysis revealed that LDB CPR was significantly associated with posterior rib fractures [odds ratio (OR) 5.37, 95% confidence interval (CI): 1.44-20.09, P = 0.01). Pneumothorax (OR 6.80, 95% CI: 0.73-62.99, P = 0.09) and the amount of pericardial fluid (OR 3.40, 95% CI: 0.20-56.32) were not significantly associated with LDB CPR. No significant difference was found for anterolateral rib fractures, sternal fractures, vertebral fractures, pleural fluid, hemothorax, hemopericardium, pneumoperitoneum, perihepatic, perisplenic and perirenal hemorrhage. CONCLUSION: Rib fractures, sternal fractures, hemothorax and hemopericardium are common CPR-related injuries. LDB CPR is significantly associated with more posterior rib fractures and a trend toward more pneumothoraces is observed when compared with manual CPR. This knowledge is important for caretakers in the case of ongoing CPR, as a pneumothorax may attribute to not achieving persistent return of spontaneous circulation, and to improve postresuscitation care of survivors.

Compound- and context-dependent effects of antibiotics on greenhouse gas emissions from livestock.

The use of antibiotics in livestock production may trigger ecosystem disservices, including increased emissions of greenhouse gases. To evaluate this, we conducted two separate animal experiments, administering two widely used antibiotic compounds (benzylpenicillin and tetracycline) to dairy cows over a 4- or 5-day period locally and/or systemically. We then recorded enteric methane production, total gas production from dung decomposing under aerobic versus anaerobic conditions, prokaryotic community composition in rumen and dung, and accompanying changes in nutrient intake, rumen fermentation, and digestibility resulting from antibiotic administration. The focal antibiotics had no detectable effect on gas emissions from enteric fermentation or dung in aerobic conditions, while they decreased total gas production from anaerobic dung. Microbiome-level effects of benzylpenicillin proved markedly different from those previously recorded for tetracycline in dung, and did not differ by the mode of administration (local or systemic). Antibiotic effects on gas production differed substantially between dung maintained under aerobic versus anaerobic conditions and between compounds. These findings demonstrate compound- and context-dependent impacts of antibiotics on methane emissions and underlying processes, and highlight the need for a global synthesis of data on agricultural antibiotic use before understanding their climatic impacts.

Dietary supplement of conjugated linoleic acids or polyunsaturated fatty acids suppressed the mobilization of body fat reserves in dairy cows at early lactation through different pathways.

To investigate the metabolic changes in the adipose tissue (AT) of dairy cows under milk fat depression (MFD), 30 cows were randomly allocated to a control diet, a conjugated linoleic acid (CLA)-supplemented diet, or a high-starch diet supplemented with a mixture of sunflower and fish oil (2:1; as HSO diet) from 1 to 112 d in milk. Performance of animals, milk yield, milk composition, energy balance, and blood metabolites were measured during lactation. Quantitative PCR analyses were conducted on the AT samples collected at wk 3 and 15 of lactation. The CLA and HSO diets considerably depressed milk fat yield and milk fat content at both wk 3 and 15 in the absence of significant changes in milk protein and lactose contents. In addition, the HSO diet lowered milk yield at wk 15 and decreased dry matter intake of cows from wk 3 to 15. Compared with the control, both CLA and HSO groups showed reduced body weight loss, improved energy balance, and decreased plasma concentrations of nonesterified fatty acids and ß-hydroxybutyrate at early lactation. The gene expression analyses reflected suppressed lipolysis in AT of the CLA and HSO groups compared with the control at wk 3, as suggested by the downregulation of hormone-sensitive lipase and fatty acid binding protein 4 and the upregulation of perilipin 2. In addition, the HSO diet promoted lipogenesis in AT at wk 15 through the upregulation of 1-acylglycerol-3-phosphate O-acyltransferase 2, mitochondrial glycerol-3-phosphate acyltransferase, perilipin 2, and peroxisome proliferator-activated receptor γ. The CLA diet likely regulated insulin sensitivity in AT as it upregulated the transcription of various genes involved in insulin signaling, inflammatory responses, and ceramide metabolism, including protein kinase B2, nuclear factor κ B1, toll-like receptor 4, caveolin 1, serine palmitoyltransferase long chain base subunit 1, and N-acylsphingosine amidohydrolase 1. In contrast, the HSO diet resulted in little or no change in the pathways relevant to insulin sensitivity. In conclusion, the CLA and HSO diets induced a shift in energy partitioning toward AT instead of mammary gland during lactation through the regulation of different pathways.

Sexual Risk Behaviors and Condom Use Barriers in Iranian Men with Substance Use Disorders.

BACKGROUND: We aimed to investigate risky sexual behaviors (RSBs) and condom use barriers in Iranian men with substance use disorders (SUDs). METHODS: Of the total 1800 outpatient drug free (ODF) and methadone maintenance treatment program (MMTP) active centers in Tehran, Iran, six were selected to participate in the current study. Data were collected (n = 300 men) using three questionnaires including a demographic questionnaire, the Risky Sexual Behavior Questionnaire (RSBQ), and the Condom Barriers Scale (CBS). The statistical software R, analysis of variance post hoc and multivariate analysis of variance (MANOVA) logistic regression tests were used in data analysis. FINDINGS: The majority, (n = 194, 64.7%) reported at least one lifetime episode of RSBs. Compared to married participants (23.1%), 88.5% of single and 87.0% of divorced men had a history of RSB. Generally, the lowest and highest subscale scores of the CBS were related to sexual experience (2.60 ± 0.71) and access/availability structure (3.77 ± 0.54), respectively. The results of MANOVA analysis showed that there was a statistically significant difference between the CSB subscales based on the participants' education and marital status (P < 0.001). Only the partner barrier subscale had a significant negative relationship (P = 0.003) with RSB. CONCLUSION: Sexual dynamic of Iranian men with SUDs is different. Barriers to condom use seem to be socio-culturally determined. Culturally acceptable strategies need to be utilized in Iranian clinical settings reaching beyond simply condom accessibility for this at risk population.

The radiodensity of cerebrospinal fluid and vitreous humor as indicator of the time since death.

PURPOSE: After death, a series of changes occur naturally in the human body in a fairly regular pattern. These postmortem changes are detectable on postmortem CT scans (PMCT) and may be useful in estimating the postmortem interval (PMI). The purpose of our study is to correlate the PMCT radiodensities of the cerebrospinal fluid (CSF) and vitreous humor (VH) to the PMI. METHODS: Three patient groups were included: group A consisted of 5 donated cadavers, group B, 100 in-hospital deceased patients, and group C, 12 out-of-hospital forensic cadavers. Group A were scanned every hour for a maximum of 36 h postmortem, and the tympanic temperature was measured prior to each scan. Groups B and C were scanned once after death (PMI range 0.2-63.8 h). Radiodensities of the VH and CSF were measured in Hounsfield units. Correlation between density and PMI was determined using linear regression and the influence of temperature was assessed by a multivariate regression model. Results from group A were validated in groups B and C. RESULTS: Group A showed increasing radiodensity of the CSF and VH over time (r (2) CSF, 0.65). PMI overruled the influence of temperature (r = 0.99 and p = 0.000). Groups B and C showed more diversity, with CSF and VH radiodensities below the mean regression line of Group A. The formula of this upper limit indicated the maximum PMI and was correct for >95 % of the cadavers. CONCLUSION: The results of group A showed a significant correlation between CSF radiodensity and PMI. The radiodensities in groups B and C were higher than in group A, therefore the maximum PMI can be estimated with the upper 95 % confidence interval of the correlation line of group A.

Compartmental flux and in situ methods underestimate total feed nitrogen as judged by the omasal sampling method due to ignoring soluble feed nitrogen flow.

The objective of the present study was to estimate ruminal feed N outflow in lactating cows using the omasal sampling, compartmental flux or in situ method. A total of five ruminally fistulated Finnish Ayrshire dairy cows were used in a 5 × 5 Latin square study with 21 d periods. Experimental silages of grass or red clover harvested at two stages of maturity in addition to a supplement of 9·0 kg concentrate/d were fed to the cows. In vivo omasal N flow was determined using the omasal sampling technique. Ruminal in situ N flow was calculated from N intake and degradability (38 µm nylon bags). The samples of ruminal contents and faeces were divided into seven particle-size fractions by wet sieving; the concentrations of indigestible neutral-detergent fibre and N were used to calculate N flow in the compartmental flux method. In vivo omasal N flow was greater for the red clover silage diets than for the grass silage diets. The N flow calculated using the compartmental flux technique and that calculated using the in situ technique were highly correlated, but both were less than and poorly correlated with the in vivo N flow. In both in situ and compartmental flux techniques, forage maturity increased the particle-associated N flow, with the increase being significantly greater for the red clover diets than for the grass silage diets. In conclusion, the compartmental flux and in situ methods described the N flow associated with the particle fractions rather than the total ruminal outflow of feed N.

Effect of DNA extraction and sample preservation method on rumen bacterial population.

The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile.