Progress 02/15/18 to 02/14/22
Outputs
Target Audience:nutritionists, dairy experts, farmers, graduate and under-graduate students, veterinarians Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?There were 3 PhD and 2 MS students along with several visiting scholars engaged in different components of research. A numbers of abstracts and posters have been presented at the ADSA annual meetings from 2018-2022. How have the results been disseminated to communities of interest?Publications, conferences, webinars, symposia. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
4. Understanding the effects of using Asparagopsis taxiformis on methanogenesis pathways and rumen microbes in the rumen.?
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2022
Citation:
DE Wasson, H Stefenoni, S Cueva, C Lage, SE R�is�nen, A Melgar, M Fetter, M Hennessy, K Narayan, N Indugu, D Pitta, C Yarish, AN Hristov. Screening of macroalgae species for enteric methane mitigation effect in vitro. Submitted to Journal of Dairy Science.
DW Pitta*, N Indugu, A Melgar, AN Hristov, K Challa, B Vecchiarelli, ML Hennessy, K Narayan, S Duval, M Kindermann, N Walker. The effect of 3-nitrooxypropanol, a potent methane inhibitor, on ruminal microbial gene expression profiles in dairy cows. Submitted to Microbiome.
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Progress 02/15/20 to 02/14/21
Outputs
Target Audience:Dairy Nutritionists, Animal Science, Microbiologists, Dairy farmers Changes/Problems:With COVID-19, we were not able to complete metatranscriptomic work for Objective 1 and 2. We anticipate completing in 2021. What opportunities for training and professional development has the project provided?Training of undergraduate and graduate students at UPenn and PSU was provided. How have the results been disseminated to communities of interest?ADSA meetings, Publications and webinars What do you plan to do during the next reporting period to accomplish the goals?Complete the metatranscriptomic work as outlined in Objectives 1 and 2.
Impacts
What was accomplished under these goals?
Objective 1 and 2 were partially accomplished and the data has been presented at the ADSA meetings in 2020.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
CFA Lage, SE Raisanen, A Melgar, K Nedelkov, X Chen, J Oh, ME Young, JS Bender, B Vecchiarelli, DW Pitta, AN Hristov (2020). Comparison of two sampling techniques for evaluating ruminal fermentation in dairy cows. Frontiers in Microbiology. 11:618032. doi: 10.3389/fmicb.2020.618032
Melgar A, Harper MT, Oh J, Giallongo F, Young ME, Ott TL, Duval S, Hristov AN. Effects of 3-nitrooxypropanol on rumen fermentation, lactational performance, and resumption of ovarian cyclicity in dairy cows. Journal of dairy science. 2020 Jan 1;103(1):410-32.
HA Stefenoni, SW R�is�nen, SF Welchez, DE Wasson, CFA Lage, A Melgar, ME Fetter, P Smith, ML Hennessy, B Vecchiarelli, J Bender, DW Pitta, CL Cantrell, C Yarish, and AN Hristov (2021). Effects of the macroalga Asparagopsis taxiformis and oregano leaves on methane emission, rumen fermentation, and lactational performance of dairy cows. Journal of Dairy Science. 104(4): 4157-4173.
DW Pitta, A Melgar, N Indugu, C Pappalardo, ML Hennessy, B Vecchiarelli, V Shabtai, M Kindermann, N Walker, AN Hristov (2021). Temporal changes in total and metabolically active ruminal methanogens induced by 3-nitrooxypropanol in dairy cows. Journal of Dairy Science (accepted).
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Progress 02/15/19 to 02/14/20
Outputs
Target Audience:Dairy Nutritionists, Animal Science, Microbiologists, Dairy farmers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?1. Graduate students were part of this project. 2. Undergraduate students worked over the summer to accomplish microbial analysis 3. Postdocs were involved in analyzing the data How have the results been disseminated to communities of interest?Results were summarized and being written up as manuscripts for publications in animal science related journals Research summaries or abstracts were accepted for presentation at ADSA meetings to be held in Florida, 2020 What do you plan to do during the next reporting period to accomplish the goals?Continue Metagenomic and Metatranscriptomic work for Objective 1 and 2.
Impacts
What was accomplished under these goals?
Objective 1: Identify differences in methanogenic pathways and determine the relationship between bacteria-archaea, hydrogen concentration, and fermentation pattern in the rumen of dairy cows with contrasting methane-yield phenotypes. One hundred thirty Holstein cows from the Pennsylvania State University dairy herd were screened for daily enteric methane (CH4) emission and CH4 yield (g/kg dry matter intake; DMI) and intensity (g/kg milk yield; MY). Five phenotypically low (LOW) and 5 phenotypically high (HIGH) CH4 emitting cows were selected for an experiment aimed at studying phenotype-related differences in methanogen diversity and rumen volatile fatty acid (VFA) profile. Cows were blocked by parity, days-in-milk, and MY. All cows were fed a standard diet for 3 wks and enteric gas emission data and rumen samples were collected during wks 4 and 5. Methane emission was measured with the GreenFeed system and rumen samples were collected by the ororuminal technique at 0, 2, 4, 8, 12, 14, 16 and 20 h post-feeding. The solid fraction from the ruminal samples were extracted for genomic DNA, PCR-amplified for archaeal 16S rDNA gene, sequenced and analyzed for archaeal diversity. The liquid fraction was used for fermentation variables. All data were analyzed using PROC MIXED of SAS. Block and block by treatment were random effects. Cows did not differ (P > 0.05) in DMI, MY or milk composition. Daily CH4 emission, yield, and intensity were lower (P ≤ 0.03) for LOW vs. HIGH cows (346 vs. 439 ± 28.6 g/d; 15.5 vs. 20.4 ± 0.92 g/kg DMI; and 10.6 vs. 13.6 ± 0.86 g/kg MY, respectively). No differences (P > 0.05) in rumen pH and total VFA concentration were observed between phenotypes. Acetate:propionate (A:P) ratio was lower (P = 0.01) in LOW vs. HIGH cows. The molar proportion of acetate was lower (P = 0.03), whereas proportion of propionate was greater (P = 0.008) in LOW cows. In this experiment, phenotypically low-CH4 yield cows had lower CH4 emission intensity, greater molar proportion of propionate and lower A:P ratio, and higher proportion of Methanosphaera vs. Methanobrevibacter in ruminal contents than phenotypically high-CH4 yield cows. Microbial analysis: The objective of this experiment was to identify differences in microbiota and methanogenesis pathways in the rumen of phenotypically low and high methane-yield cows. Following an initial screening period, 5 phenotypically low (LOW) and 5 phenotypically high (HIGH) methane-emitting cows, blocked by parity, days in milk, and milk yield, were used to investigate the functional role of rumen microbiota. All cows were adapted to a standard diet for 3 weeks and sampled for rumen contents at 0, 2, 4, 8, 12, 14, 16, and 20 h post-feeding using the ororuminal technique. The solid fraction of the ruminal samples was extracted for DNA, PCR-amplified for the bacteria and archaea 16S rDNA gene, sequenced and analyzed for bacteria and archaeal diversity. Additionally, the genomic DNA from 2h post-fed rumen samples was prepared for metagenomic analysis and genes were annotated against the KEGG database. Methane emissions (346 vs 439 ± 28.6 g/d) were lower (P = 0.03) for LOW vs. HIGH cows. At the community level, both unweighted and weighted UniFrac analysis revealed differences (P < 0.05) in bacteria and archaea communities between groups. Among methanogens, Methanobrevibacter was lower and Methanosphaera was higher (P < 0.05 for both) in LOW vs. HIGH groups. In bacterial DNA and cDNA, genera such as Prevotellaceae and Succinovibrionaceae were higher in the LOW group, while genera such as Ruminococcaceae were higher in the HIGH group. Among the methanogenesis pathways, across both groups, the CO2/formate-reducing pathway was predominant followed by the methanol-reducing pathway. Methanobrevibacter and Methanosphaera, respectively, played significant roles in these pathways. The number of gene copies encoding the alpha, beta, and gamma chains of methyl co-enzyme reductase (MCR), an enzyme present in all methanogens that facilitates the formation of methane, was lower (P <0.05) in LOW vs. HIGH cows (336 vs. 452 copies per million, respectively). It can be concluded that the 22% difference in methane emissions is accompanied by a 25% difference in gene copies coding for MCR enzyme between HIGH and LOW cows. Objective 2: Determine the extent to which methane inhibitors can affect rumen microbiota, rumen fermentation, and dairy cow productivity. The objective of this experiment was to determine the effect of Asparagopsis taxiformis (AT) and oregano leaves (ORE) on enteric methane (CH4) emission, rumen fermentation, and lactational performance of dairy cows. Twenty Holstein cows (± SD) [95 ± 22.0 days in milk and 42 ± 2.6 kg milk yield (MY)] were used in a replicated 4 × 4 Latin square design with 4, 28-d periods. Treatments were a basal diet (CON) and CON supplemented with 0.25% AT (LAT), 0.50% AT (HAT), or 2.0% ORE of dry matter intake (DMI). Enteric gas emissions were measured using the GreenFeed system and rumen samples were collected by the ororuminal technique. Data were analyzed using PROC MIXED of SAS with treatment and period in the model. Square and cow within square were random effects. Compared with CON, HAT decreased (P < 0.001) average daily CH4 emission and CH4 yield by 65% (380 and 131 g/d) and 55% (14.0 and 6.32 g/kg DMI), respectively, in experimental periods (P)1 and 2, but had no effect in P3 and P4. The differential response to HAT in P3 and 4 could be attributed to a decrease in bromoform concentration in AT over time (about 74% decrease in 5 mo of storage) observed in a separate experiment. ORE and LAT had no effect on CH4 emission. Compared with CON, HAT decreased (P ≤ 0.006) DMI, MY, and energy-corrected MY by 6.9, 5.7, and 7.3%, respectively. Milk fat and true protein concentrations were not affected by treatment, but lactose was decreased (P < 0.001) by HAT, compared with CON. Total VFA concentration and acetate:propionate ratio tended (P = 0.06) to be lowest for HAT. HAT and ORE increased (P = 0.02) the molar proportion of propionate compared with CON and LAT. Both AT treatments had greater concentrations of butyrate (P < 0.001) compared with ORE and CON. In this experiment, AT fed at 0.5% of DMI decreased CH4 emission by ≥ 55% in P1 and P2 of the experiment but the effect disappeared by P3 and P4, most likely due to a decrease in bromoform concentration. HAT also decreased DMI and milk production. ORE had no effect on CH4 emission and lactational performance of the cows. Towards the end of each period, rumen contents were sampled and was extracted for DNA, PCR-amplified for the 16S rDNA gene of bacteria and archaea, sequenced and analyzed for bacteria and archaea diversity. The enteric methane emission data showed a decrease in the mitigation effect of AT over time (a 52-57% reduction in methane yield in experimental periods 1 and 2 for HAT and no statistical effect in P3 and P4). At the community level, both bacterial and archaeal communities were different (P < 0.05) between treatments. Methanosphaera differed between HAT and control with a significant reduction for HAT in P1, a smaller decrease in P2, and no difference from CON in P3 and P4, whereas Methanobrevibacter populations were unaltered. Butyrate-producing bacteria such as Butyrivibrio, Moryella, and Eubacterium were higher in HAT treatments compared with CON and ORE throughout the experiment. These findings indicate that AT selectively inhibits specific clades of methanogens such as Methanosphaera, which may have a greater share in total methane formation in the rumen than previously thought. Inhibition of methanogens was accompanied by significant alterations in the rumen bacterial populations revealing that alternate pathways of fermentation may be triggered with methane inhibition.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
DW Pitta, HA Stefenoni, N Indugu, M Hennessy, B Vecchiarelli, R Shah, S Garapati, C Yarish, SC Welchez, SE R�is�nen, DE Wasson, C Lage, A Melgar, ME Fetter, and AN Hristov. The effects of supplementing a seaweed, Asparagopsis taxiformis, to dairy cows on bacteria-archaea interactions in the rumen. In ADSA Annual Meetings June 21-24, 2020, West Palm Beach, Florida, USA
HA Stefenoni, SC Welchez, SE Raeisaenen, DE Wasson, A Melgar, ME Fetter, CFA Lage, M Hennessey, N Indugu, DW Pitta, and AN Hristov. Differences in rumen volatile fatty acid profile and archaeal diversity in low- and high-enteric methane yield phenotype dairy cows. In ADSA Annual Meetings June 21-24, 2020, West Palm Beach, Florida, USA
DW Pitta, HA Stefenoni, N Indugu, M Hennessy, B Vecchiarelli, V Shabtai, SC Welchez, SE R�is�nen, DE Wasson, A Melgar, ME Fetter, and AN Hristov. Differences in methanogenesis pathways and microbial diversity in the rumen of low and high methane-yield phenotype dairy cows In ADSA Annual Meetings June 21-24, 2020, West Palm Beach, Florida, USA
HA Stefenoni, SE R�is�nen, SF Welchez, DE Wasson, CFA Lage, A Melgar, ME Fetter, M Hennessy, B Vecchiarelli, J Bender, DW Pitta, C Yarish, and AN Hristov. Effects of Asparagopsis taxiformis and oregano leaves on methane emission, rumen fermentation, and lactational performance of dairy cows. In ADSA Annual Meetings June 21-24, 2020, West Palm Beach, Florida, USA
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Progress 02/15/18 to 02/14/19
Outputs
Target Audience:Target Audience: Dairy Nutritionists, Veterinarians, animal scientists, post-graduates, vet-technicians Changes/Problems:Considered factors such as age, time of entry into lactation, it is not possible to cannulate the selected cows. We rely on our conclusion that there were no differences between tube and cannula samples for microbial analysis and molar proportions of VFA and have decided to use intact animals for the animal experiment. We anticipate using intact animals will help us to perform animal trials in a timely manner with greater accuracy in microbial and enteric methane data. ? What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Findings of this project will be presented at the ADSA meetings to be held at Cinncinnati, Ohio. The data was summarized and submitted as abstracts. These are accepted for publications. Further, two manuscripts are currently in preparation. What do you plan to do during the next reporting period to accomplish the goals?We propose to perform animal experiments in Fall of 2019
Impacts
What was accomplished under these goals?
For objective 3, six cows were enrolled in the study to investigate if saliva, bolus, stomach tube (ST) and feces may be a proxy for rumen cannula samples (CS). The experiment was conducted in May 2018. All cows were sampled for saliva, bolus, tube, cannula and feces at prefeed and 2h, 4h, 6h, 8h, and 12h post feeding. The CS and ST samples were further fractionated to whole, solid and liquid samples. Samples were collected for DNA and RNA separately for microbial analysis. The liquid fraction from CS and ST samples were analyzed to fermentation parameters (pH, VFA, dissolved hydrogen and protozoal counts). Intake, milk weights and components were also recorded for these cows. Conclusions from microbial analysis: Both saliva and feces had certain bacteria that were not previously reported in the rumen, suggesting these sample types have distinct microbial fingerprints. Notably, bolus samples at 6, 8 and 12 h had similar compositions with the corresponding cannula samples. There were several bacterial lineages in bolus samples also detected in saliva samples, but not in other sample types, indicating possible contamination of the bolus sample with saliva. It can be concluded the ST method may serve as a proxy to CS for microbial investigations. Bolus samples may also serve as a proxy, however, sampling time and removal of bacterial contaminants from saliva will be needed for microbial analysis. The archaea diversity from the above samples is currently being analyzed. The RNA from tube and cannula sample samples were processed for bacterial analysis subsequently. The metabolically active bacterial populations in the rumen were different to the total bacterial populations in the same sample. There were no differences between tube and cannula derived metabolically active bacterial communities indicating samples collected using stomach tube method may serve as proxy for bacterial analysis. There were small changes noted at the individual bacterial taxa between stomach and tube samples. Conclusions from fermentation analysis of stomach tube (ST) and cannula sample (CS): Compared with CS, ST samples had or tended to have greater (P < 0.01) pH (average 6.26 and 6.73, respectively), lower (P < 0.01) protozoal counts (average 26.7 x 104 and 8.70 × 104, respectively) and lower total (average 142 and 109 mM, respectively) and individual volatile fatty acid (VFA) concentrations (P < 0.01). Molar proportions of individual VFA tended to be lower (P ≤ 0.07) for ST compared with CS, except for butyrate and propionate, which did not differ between methods (P ≥ 0.17). It was inferred that rumen samples collected vis ST have lower VFA concentrations and protozoa counts when compare to CS method; however, the molar proportions of VFAs were similar between the two methods. For objective 1 and 2, we have screened 96 cows for enteric methane emitting potential. A selected cohort of cows representing High- and Low emitters for enteric methane (methane/Kg DM intake) will be enrolled for this study. The animal experiment will be conducted in Fall of 2019. Considered factors such as age, time of entry into lactation, it is not possible to cannulate the selected cows. We rely on our conclusion that there were no differences between tube and cannula samples for microbial analysis and molar proportions of VFA and have decided to use intact animals for the animal experiment. We anticipate using intact animals will help us to perform animal trials in a timely manner with greater accuracy in microbial and enteric methane data.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
1. DW Pitta, CFA Lage, SE R�is�nen, A Melgar, K Nedelkov, X Chen, J Oh, N Indugu, B Vecchiarelli, J Bender, AN Hristov. 2019. Comparison of non-invasive ruminal sampling techniques to that of standard cannula method for ruminal microbial analysis. In ADSA Annual Meetings. June 23-26, 2019, Cincinnati, Ohio, USA
2. CFA Lage, SE R�is�nen, A Melgar, K Nedelkov, X Chen, J Oh, B Vecchiarelli, J Bender, DW Pitta, AN Hristov. 2019. Comparison of two sampling techniques for evaluating ruminal fermentation in dairy cows. In ADSA Annual Meetings. June 23-26, 2019, Cincinnati, Ohio, USA
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