scholarly journals Changes in the Rumen Bacteriome Structure and Enzymatic Activities of Goats in Response to Dietary Supplementation with Schizochytrium spp.

2021 ◽  
Vol 9 (7) ◽  
pp. 1528
Author(s):  
Alexandros Mavrommatis ◽  
Dimitrios Skliros ◽  
Emmanouil Flemetakis ◽  
Eleni Tsiplakou
Keyword(s):  
Dairy Products ◽  
Rumen Fluid ◽  
Cellulase Activity ◽  
Control Group ◽  
Cellulolytic Bacteria ◽  
Sequencing Platform ◽  
Rumen Microbiome ◽  
Nutritional Strategy ◽  
C 50 ◽  
Treponema Bryantii

With the aim to produce functional dairy products enriched with polyunsaturated fatty acids (PUFA) by using feed supplements, radical changes could occur in the rumen microbiome. This work investigated the alterations of the rumen bacteriome of goats fed with PUFA-rich marine microalgae Schizochytrium spp. For the trial, twenty-four goats were divided into four homogenous clusters (six goats/treatment) according to their fat-corrected (4%) milk yield, body weight, and age; they were individually fed with alfalfa hay and a concentrate (F/C = 50/50). The concentrate of the control group (CON) contained no microalgae, while those of the treated groups were supplemented daily with 20 (ALG20), 40 (ALG40), and 60 g (ALG60) of Schizochytrium spp./goat. Rumen fluid samples were collected using a stomach tube during the 20th and 40th days of the experiment. The microbiome analysis using a 16S rRNA sequencing platform revealed that Firmicutes were decreased in microalgae-fed goats, while Bacteroidetes showed a tendency to increase in the ALG40 group due to the enhancement of Prevotellaceae. Cellulolytic bacteria, namely Treponema bryantii, Ruminococcus gauvreauii, R. albus, and R. flavefaciens, were decreased in the ALG40 group, resulting in an overall decrease of cellulase activity. In contrast, the amylolytic potential was significantly enhanced due to an upsurge in Ruminobacter amylophilus, Succinivibrio dextrinosolvens, and Fretibacterium fastidiosum populations. In conclusion, supplementing goats’ diets with 20 g Schizochytrium spp. could be considered a sustainable and efficient nutritional strategy to modulate rumen microbiome towards the development of dairy products enriched with bioactive compounds, while higher levels induced substantial shifts in determinant microbes’ populations.

scholarly journals Alterations in the Rumen Particle-Associated Microbiota of Goats in Response to Dietary Supplementation Levels of Schizochytrium spp.

2021 ◽  
Vol 13 (2) ◽  
pp. 607
Author(s):  
Alexandros Mavrommatis ◽  
Dimitrios Skliros ◽  
Marica Simoni ◽  
Federico Righi ◽  
Emmanouil Flemetakis ◽  
...  
Keyword(s):  
Nutritional Value ◽  
Dietary Supplementation ◽  
Significant Decline ◽  
Rumen Content ◽  
Control Group ◽  
Rumen Microbiome ◽  
Nutritional Strategy ◽  
Relative Abundances ◽  
C 50

Fat rich microorganisms, such as microalgae Schizochytrium spp., are potential biotechnological tools in the modulation of rumen microbiome towards ecofriendly and high nutritional value end-products. However, limited in vivo trials have been reported on the topic. The aim of this study was to contribute to the knowledge on the effect of fat rich microalgae on the methanogenic and feed degrading particle-associated microbes in goats’ rumen content. For the trial, twenty-four goats were divided into four homogenous clusters (six goats/treatment) according to their fat corrected (4%) milk yield, body weight and age and individually were fed with alfalfa hay and concentrate feeds (F/C = 50/50). The concentrate of the control group (CON) contained no microalgae, while those of the treated groups were supplemented daily with 20 (ALG20), 40 (ALG40), and 60 (ALG60) g of Schizochytrium spp./goat. The relative abundances of total Archaea, methanogens, Methanomassiliicoccales, Methanobrevibacter spp., Methanosphaera stadmanae and Methanobacterium formicicum were significantly (p < 0.05) decreased in microalgae-fed goats compared to the CON ones. Moreover, a significant decline in the relative abundances of Firmicutes, Ruminococcus flavefaciens, Butyrivibrio fibrosolvents, and Neocallimastigales in the rumen particle-associated microbiota of microalgae supplemented goats were observed. In conclusion, goats’ diets supplementation with Schizochytrium spp., could be considered a sustainable nutritional strategy for methanogens inhibition in their rumen particle-associated microbiota.

scholarly journals Correlation of the rumen fluid microbiome and the average daily gain with a dietary supplementation of Allium mongolicum Regel extracts in sheep1

2019 ◽  
Vol 97 (7) ◽  
pp. 2865-2877 ◽  
Author(s):  
Hongxi Du ◽  
Khas Erdene ◽  
Shengyang Chen ◽  
Saruli Qi ◽  
Zhibi Bao ◽  
...  
Keyword(s):  
Rumen Fluid ◽  
Average Daily Gain ◽  
Basal Diet ◽  
Experimental Period ◽  
Control Group ◽  
Illumina Hiseq ◽  
Rumen Microbiome ◽  
Microbiome Profile ◽  
Dietary Treatments ◽  
Powder Extract

Abstract Plant extracts can affect the rumen microbiome and ADG in ruminants, and studies of the association between the rumen microbiome and ADG provide information applicable to improving ruminant growth performance. The objectives were to investigate the effects of Allium mongolicum Regel extracts on the rumen microbiome and ADG and their association in sheep. Forty healthy, male, small-tailed Han sheep (6 mo, 34 ± 3.5 kg body weight) were randomly assigned to 1 of the following 4 dietary treatments: basal diet as control group (CK, n = 10), basal diet supplemented with 3.4 g·sheep−1·d−1A. mongolicum Regel powder extract as PAM group (PAM, n = 10), basal diet supplemented with 10 g·sheep−1·d−1A. mongolicum Regel powder as AM group (AM, n = 10), and basal diet supplemented with 10 g·sheep−1·d−1A. mongolicum Regel powder extract residue as RAM group (RAM, n = 10). The ADG for individual sheep was calculated using the sum of the ADGs observed during the experimental period divided by the number of days in the experimental period. At the end of the experiment, sheep were randomly selected from each treatment for slaughter (n = 6), and the rumen fluids were collected and stored immediately at −80 °C. Illumina HiSeq was subsequently used to investigate the changes in the rumen microbiome profile, and the associations with ADG were analyzed by Spearman correlation coefficient analysis. The results demonstrated that, compared with that in CK group, the ADG in AM and RAM significantly increased (P = 0.0171). The abundances of Tenericutes and Mollicutes ([ρ] = 0.5021, P = 0.0124) were positively correlated with ADG. Within Mollicutes, the abundances of Anaeroplasmatales ([ρ] = 0.5458, P = 0.0058) and Anaeroplasmataceae ([ρ] = 0.5458, P = 0.0058) were positively correlated with ADG. The main negatively correlated bacteria were Saccharibacteria ([ρ] = −0.4762, P = 0.0187) and Betaproteobacteria ([ρ] = −0.5669, P = 0.039). Although Anaeroplasmatales and Anaeroplasmataceae were positively correlated with ADG, Saccharibacteria and Betaproteobacteria were negatively correlated with ADG. In conclusion, supplementation with A. mongolicum Regel powder and extracts will influence the rumen microbiome and increase the ADG.

scholarly journals Autism Spectrum Disorder (ASD) with and without Mental Regression is Associated with Changes in the Fecal Microbiota

Nutrients ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 337 ◽  
Author(s):  
Julio Plaza-Díaz ◽  
Antonio Gómez-Fernández ◽  
Natalia Chueca ◽  
María Torre-Aguilar ◽  
Ángel Gil ◽  
...  
Keyword(s):  
Intestinal Microbiota ◽  
Present Report ◽  
Principal Component ◽  
Autism Spectrum ◽  
Control Group ◽  
Healthy Group ◽  
Sequencing Platform ◽  
Preliminary Results ◽  
Children With Asd ◽  
Mental Regression

New microbiome sequencing technologies provide novel information about the potential interactions among intestinal microorganisms and the host in some neuropathologies as autism spectrum disorders (ASD). The microbiota–gut–brain axis is an emerging aspect in the generation of autistic behaviors; evidence from animal models suggests that intestinal microbial shifts may produce changes fitting the clinical picture of autism. The aim of the present study was to evaluate the fecal metagenomic profiles in children with ASD and compare them with healthy participants. This comparison allows us to ascertain how mental regression (an important variable in ASD) could influence the intestinal microbiota profile. For this reason, a subclassification in children with ASD by mental regression (AMR) and no mental regression (ANMR) phenotype was performed. The present report was a descriptive observational study. Forty-eight children aged 2–6 years with ASD were included: 30 with ANMR and 18 with AMR. In addition, a control group of 57 normally developing children was selected and matched to the ASD group by sex and age. Fecal samples were analyzed with a metagenomic approach using a next-generation sequencing platform. Several differences between children with ASD, compared with the healthy group, were detected. Namely, Actinobacteria and Proteobacteria at phylum level, as well as, Actinobacteria, Bacilli, Erysipelotrichi, and Gammaproteobacteria at class level were found at higher proportions in children with ASD. Additionally, Proteobacteria levels showed to be augmented exclusively in AMR children. Preliminary results, using a principal component analysis, showed differential patterns in children with ASD, ANMR and AMR, compared to healthy group, both for intestinal microbiota and food patterns. In this study, we report, higher levels of Actinobacteria, Proteobacteria and Bacilli, aside from Erysipelotrichi, and Gammaproteobacteria in children with ASD compared to healthy group. Furthermore, AMR children exhibited higher levels of Proteobacteria. Further analysis using these preliminary results and mixing metagenomic and other “omic” technologies are needed in larger cohorts of children with ASD to confirm these intestinal microbiota changes.

scholarly journals PSVI-22 Rumen microbiome of beef cattle is modulated by backgrounding systems

2020 ◽  
Vol 98 (Supplement_3) ◽  
pp. 220-220
Author(s):  
Bobwealth O Omontese ◽  
Ashok K Sharma ◽  
Jason Langlie ◽  
Joe Armstrong ◽  
Alfredo DiCostanzo ◽  
...  
Keyword(s):  
Beef Cattle ◽  
Developmental Stages ◽  
Production Systems ◽  
Rumen Fluid ◽  
Illumina Miseq ◽  
High Energy ◽  
Shannon Index ◽  
Bacterial Gene ◽  
Beef Calves ◽  
Rumen Microbiome

Abstract Backgrounding (BKG) segment in beef production systems is characterized by utilization of different forages which affect growth performance and carcass characteristics. However, it is unclear how BKG systems impact rumen microbiome. We investigated rumen microbiome dynamics of beef calves under different BKG systems. At weaning, Angus and Angus x Simmental beef calves (n = 38) were stratified by age, weight, and sex in a completely randomized design into 1 of 3 BKG treatments for 55 d: 1) perennial pasture (PP; quackgrass, orchardgrass; smooth bromegrass, red clover, and alfalfa); 2) summer annual cover crop (CC; cereal oats, purple top turnips, hunter forage brassica, and graza forage radish); and 3) dry lot (DL; haylage, corn, and DDGS). After BKG, all calves were assigned to a high energy ration in a feedlot. Rumen sample was collected via esophageal tubing at weaning, BKG and feedlot. A total of 190 rumen fluid samples were used to sequence the hypervariable V4 region of the 16S rRNA bacterial gene on an Illumina MiSeq platform. The results showed that BKG systems largely influenced rumen bacterial communities. Specifically, microbiome composition and diversity were not different at weaning, diverged significantly during BKG (Shannon index, Bray Curtis distance metrics; P &lt; 0.001) and homogenized during feedlot. During the BKG segment, the bacterial genera Agrobacterium, Coprococcus, and Ruminococcus were dominant in CC whereas Fibrobacteraceae and Mycoplasmataceae was most dominant in DL. Moreover, rumen microbiome patterns of CC and DL calves showed increased plasticity in early stages of development but not during feedlot with PP showing fewer changes over time. These results indicate that BKG systems significantly modulate the rumen microbiome of beef cattle and, underscore the importance of early developmental stages as potential targets for feeding interventions that can impact the animal microbiome to enhance animal performance.

scholarly journals Effects of Saccharomyces cerevisiae Culture on Ruminal Fermentation, Blood Metabolism, and Performance of High-Yield Dairy Cows

Animals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 2401
Author(s):  
Xiaoge Sun ◽  
Yue Wang ◽  
Erdan Wang ◽  
Shu Zhang ◽  
Qianqian Wang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dairy Cows ◽  
Milk Yield ◽  
Inflammatory Cytokines ◽  
Rumen Fluid ◽  
Fat Content ◽  
High Yield ◽  
Control Group ◽  
And Performance ◽  
Basal Ration

High-yield dairy cows with high-concentrate diets are more prone to experiencing health problems associated with rumen microbial imbalance. This study assessed the effects of Saccharomyces cerevisiae culture (SC), a food supplement, on ruminal pH, volatile fatty acid (VFA), inflammatory cytokines, and performance of high-yield dairy cows. Forty Holstein cows with similar characteristics (e.g., milk yield, days of milk, and parity) were randomly divided into two groups: an experimental group fed the basal ration supplemented with the SC of 100 g of SC per cow per day (hour, SC group), and a control group fed the same basal ration diet without SC (i.e., CON group). On average, the supplementation of SC started at 73 days of lactation. The experimental period lasted approximately 70 days (from 18 January to 27 March 2020), including 10 days for dietary adaptation. Milk yield was recorded daily. Rumen fluid and milk samples were collected after 2 h of feeding in the morning of day 0, 15, 30, and 60. The data showed that rumen pH increased (p < 0.05) when cows were provided with SC. On average, the cows in the SC group produced 1.36 kg (p < 0.05) more milk per day than those in the CON group. Milk fat content of cows in the SC group was also higher (4.11% vs. 3.96%) (p < 0.05). Compared with the CON group, the concentration of acetic acid in the rumen fluid of dairy cows in the SC group was significantly higher (p < 0.05). There were no differences (p > 0.05) found in milk protein content and propionic acid between groups. The SC group had a tendency increase in butyric acid (p = 0.062) and total VFA (p = 0.058). The result showed that SC supplementation also enhanced the ratio between acetic and propionic. Most of the mean inflammatory cytokine (IL-2, IL-6, γ-IFN, and TNF-α) concentrations (p < 0.05) of the SC group were lower than CON group. This study demonstrated that high-yield cows receiving supplemental SC could produce more milk with higher fat content, have higher rumen acetate, and potentially less inflammatory cytokines.

Milk productivity of cows, quality of milk and dairy products when using natural feed additive

2020 ◽  
pp. 13-21
Author(s):  
Yu. A. Karmatskikh ◽  
N. M. Kostomakhin
Keyword(s):  
Milk Production ◽  
Dairy Products ◽  
Feed Additive ◽  
Control Group ◽  
Black And White ◽  
Mineral Additive ◽  
Quality Of Milk ◽  
Experimental Group ◽  
Milk And Dairy Products

The organization of feeding of highly productive cows is one of the most difficult tasks in dairy cattle breeding. The purpose of the work was to study the influence of natural mineral additive on the productivity, composition and some technological qualities of milk of cows of Black-and-White breed of Ural type. Research and production experiment has been carried out in CJSC “Glinki” in the Ketovsky district in the Kurgan region on Black-and-White cows. The influence of bentonite additive in the ration of highly productive cows during the period of increasing the milk yield on the composition and technological quality of milk and dairy products has been determined. In general, for the first 100 days of lactation the cows of the experimental group have produced by 191 kg more natural fat milk or 8,4 %, compared to the control group. In terms of 4 % milk from animals of the experimental group has been got by 233 kg or 9,3 % more than of the control group. The yield of milk fat in the experimental group was by 10,44 kg or 12,9 % more than the control group. The difference in milk protein yield for 100 days of lactation was 7,34 kg or 9,8 %. The economic indicators (prime-cost, profitability) of milk production when feeding the mineral additive bentonite to cows have been calculated. With an increase in the total cost of obtaining milk from cows of the experimental group, caused by higher milk yields and the purchase of bentonite, the revenue from the sale of milk from this group was by 7,83 thousand rubles or 8,4 % more. As a result, milk production from animals of the experimental group was by 3,44 % more efficient. It has been found that the inclusion of bentonite in the composition of concentrated feed in the amount of 400 g/head/day allowed to reduce their expenditure, as well as EFU and digestible protein, which ultimately provided a higher level of profitability of milk production.

PSVII-14 Feeding carbohydrases to grazing beef cattle under different nutritional strategies on dry season: Ruminal fermentation

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 449-450
Author(s):  
Anderson Acosta ◽  
Guilherme S Vasconcellos ◽  
Alexandre Perdigão ◽  
Victor Valério de Carvalho ◽  
Tiago S Acedo ◽  
...  
Keyword(s):  
Rumen Fluid ◽  
Latin Square ◽  
Experimental Period ◽  
Dry Season ◽  
Ruminal Fermentation ◽  
Beta Glucanase ◽  
Randomized Design ◽  
Nutritional Strategy ◽  
Completely Randomized Design ◽  
Nutritional Strategies

Abstract The aim with this trial was to evaluate two nutritional plans with the addition of carbohydrases enzyme blend (CEB) on ruminal fermentation of grazing Nellore bulls in dry season. Five rumen cannulated Nellore steers (BW = 350 kg) were used. Animals were randomly assigned to a treatment sequence in a 5×5 Latin square experiment with 15 d-periods (10 d to diet adaptation and 5 d for data collection), following a completely randomized design, being the treatments: mineral-proteic supplementation (Fosbovi® Proteico 35; PS) offered at 0,1% BW (0 or 4.75 g/animal/day of CEB) or mineral-protein-energetic supplementation (Fosbovi® Proteico-Energético 25; PES) offered at 0.3% BW (0, 4.75 or 9.50 g/animal/day of CEB). The CEB was mainly composed by beta-glucanase and xylanase enzymes (Ronozyme® VP and Ronozyme® WX). Both enzymes and supplements were provided by DSM Nutritional Products Brasil S.A. Rumen fluid samples were collected on the last day of each experimental period before (0), and 2, 4, 6, and 8 h after supplements were offered. Data were analyzed using PROC MIXED of SAS. Steers fed PSE had greater (P ≤ 0.041) rumen concentration of acetate, propionate, and butyrate, and lower (P = 0.028) acetate to propionate ratio in comparison with animals fed PS. Rumen acetate concentrations were 61.6 and 58.8 mmol/L and rumen propionate concentrations were 15.2 and 13.7 mmol/L for animals under the PSE and PS, respectively. A quadratic effect (P = 0.048) for carbohydrases enzyme blend was observed in NH3-N ruminal concentration of PES animals, where 4.75 g/day level presented the lower concentrations compared to control (24.13 vs. 32.20 mg/dL). Furthermore, rumen concentrations of acetate (P = 0.002) and propionate (P = 0.021) linearly increased with enzymes inclusion for PSE plan. PSE nutritional strategy positively influence ruminal fermentation of Nellore steers in dry season and its effects can be enhanced with CEB inclusion of 4.75 g/day.

scholarly journals Non-structural carbohydrates in the nutrition of high-yielding dairy cows during a transition period

2010 ◽  
Vol 55 (No. 11) ◽  
pp. 468-478 ◽  
Author(s):  
K. Poláková ◽  
V. Kudrna ◽  
A. Kodeš ◽  
B. Hučko ◽  
Z. Mudřík
Keyword(s):  
Dairy Cows ◽  
Milk Yield ◽  
Body Condition ◽  
Dry Matter ◽  
Rumen Fluid ◽  
Live Weight ◽  
Dry Matter Intake ◽  
Control Group ◽  
Maize Starch ◽  
Milk Performance

The main aim of this study was to investigate experimentally the effect of different composition of non-structural carbohydrates (NFC) in prepartum feed rations administered to high-yielding dairy cows at a high concentration of NFC in the diet on dry matter intake both before and after parturition and on subsequent milk performance, body condition and physiological traits of rumen fluid and blood. Thirty-six high-yielding dairy cows were allocated into one of the three well-balanced groups (K, O, and C), and each group received a different feeding rations. Feeding rations differed in non-structural carbohydrate (NFC) structure. The "K" (control) group received a feeding ration with NFC in the form of maize starch in particular, while the feeding rations of the other two (experimental) groups contained either (besides maize starch) saccharose from dried sugar beet (the "O" group) or a dominant amount of NFC was in the form of saccharose (the "C" group). After calving, all dairy cows were given the same feeding ration from the first day after parturition. The experiment was conducted for 21 days before and 50 days after calving. FR in the form of total mixed ration was offered ad libitum. Dry matter intake, milk performance, body condition, live weight, and blood and rumen parameters were recorded for the duration of the experiment. Average daily dry matter intake before calving was highest in the "K" group (14.32 kg per head). Differences among groups were statistically significant (P &lt; 0.05). Prepartum dry matter consumption dropped as the rate of saccharose in the diet of cows increased. Dry matter consumption levelled off after calving. Milk yield was also highest in the "K" group (43.71 kg/head/day), but fatness of milk and thus the production of fat corrected milk were lowest in this group. The highest milk fat content (4.10%) and fat corrected milk production (44.03 kg/head/day) were recorded in the "C" group, whereas the highest milk protein concentration was found in the milk of the "O" group. The composition of NFC affected dry matter intake before parturition, but these concentrations did not significantly affect dry matter intake, milk yield, milk composition, live weight, body condition or blood serum and rumen fluid parameters after calving

scholarly journals Dynamics and stabilization of the rumen microbiome in yearling Tibetan sheep

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Lei Wang ◽  
Ke Zhang ◽  
Chenguang Zhang ◽  
Yuzhe Feng ◽  
Xiaowei Zhang ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Rumen Fluid ◽  
Management Strategies ◽  
Rrna Gene ◽  
Functional Development ◽  
Rumen Microbiome ◽  
Age Related ◽  
Colonization Process ◽  
Rrna Gene Sequencing ◽  
First Year Of Life

AbstractThe productivity of ruminants depends largely on rumen microbiota. However, there are few studies on the age-related succession of rumen microbial communities in grazing lambs. Here, we conducted 16 s rRNA gene sequencing for bacterial identification on rumen fluid samples from 27 Tibetan lambs at nine developmental stages (days (D) 0, 2, 7, 14, 28, 42, 56, 70, and 360, n = 3). We observed that Bacteroidetes and Proteobacteria populations were significantly changed during the growing lambs’ first year of life. Bacteroidetes abundance increased from 18.9% on D0 to 53.9% on D360. On the other hand, Proteobacteria abundance decreased significantly from 40.8% on D0 to 5.9% on D360. Prevotella_1 established an absolute advantage in the rumen after 7 days of age. The co-occurrence network showed that the different microbial of the rumen presented a complex synergistic and cumbersome relationship. A phylogenetic tree was constructed, indicating that during the colonization process, may occur a phenomenon in which bacteria with close kinship are preferentially colonized. Overall, this study provides new insights into the colonization of bacterial communities in lambs that will benefit the development of management strategies to promote colonization of target communities to improve functional development.

Interactions between Treponema bryantii and cellulolytic bacteria in the in vitro degradation of straw cellulose

1987 ◽  
Vol 33 (3) ◽  
pp. 244-248 ◽  
Author(s):  
H. Kudo ◽  
K.-J. Cheng ◽  
J. W. Costerton
Keyword(s):  
Plant Cell Wall ◽  
Bacterial Species ◽  
Barley Straw ◽  
Cellulolytic Bacteria ◽  
Morphological Examination ◽  
Cellulose Digestion ◽  
Acid Generation ◽  
Pure Cultures ◽  
Treponema Bryantii

To assess the contribution of individual bacterial species to the overall process of cellulose digestion in the rumen, cellulolytic bacteria (Bacteroides succinogenes and Ruminococcus albus) were tested as pure cultures and as cocultures with noncellulolytic Treponema bryantii. In studies of in vitro barley straw digestion, Treponema cocultures surpassed pure cultures of the cellulolytic organisms in dry matter disappearance, volatile fatty acid generation, and in the production of succinic acid, lactic acid, and ethanol. Morphological examination, by electron microscopy, showed that cells of T. bryantii associate with the plant cell wall materials in straw, but that cellulose digestion occurs only when these organisms are present with cellulolytic species such as B. succinogenes. These results show that cellulolytic bacteria interact with noncellulolytic Treponema to promote the digestion of cellulosic materials.

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