scholarly journals Effects of Fermented Oat Straw as a Lovastatin Carrier on in vitro Methane Production and Rumen Microbiota

2021 ◽  
Vol 9 ◽  
Author(s):  
Amaury Ábrego-Gacía ◽  
Héctor M. Poggi-Varaldo ◽  
Alfredo Mendoza-Vargas ◽  
Francisco G. Mercado-Valle ◽  
Elvira Ríos-Leal ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Statistical Significance ◽  
Specific Inhibitor ◽  
Control Treatment ◽  
Molar Ratio ◽  
Ruminal Fermentation ◽  
Rumen Microbiota ◽  
Alpha And Beta Diversity ◽  
Oat Straw

To date, there is an urgent need for implementing practical strategies to reduce CH4 emissions from ruminants. Lovastatin (Lv) is a specific inhibitor of methanogenic archaea. Due to the high cost of pure Lv, solid-state fermentation might be an economical bioprocess to produce Lv and facilitate its use in ruminant nutrition. The goal of this work was to assess the effects of supplementing fermented oat straw as a lovastatin carrier (FOS) to a high-grain ration on in vitro CH4 inhibition and rumen microbiota in beef cattle. The experimental design of in vitro rumen fermentation was completely randomized with four concentrations of Lv in the diet mixture. The supplementation with FOS to give Lv concentration of 100 and 150 mg L−1 in the ruminal fermentation medium significantly inhibited methanogenesis at similar levels. This suggested that less than 20% of FOS was required in the ration to achieve up to 38% of CH4 mitigation without affecting the chemical composition and nutritional value of the ration. Short-chain fatty acid (SCFA) production and profile showed that only the treatments with Lv at 100 and 150 mg L−1 decreased the concentration of total SCFAs; the molar ratio of propionate significantly increased with respect to that of the control. Treatment with Lv at 150 mg L−1 did not result in significant differences in the alpha and beta diversity indices compared to the control. However, significant changes in the relative abundance of some microorganisms were detected, such as an increase in Ruminococcus and a decrease in Prevotella. The predominant 99%+ MA in all controls, treatment, and inocula samples belonged to the Methanobrevibacter genus and very small (negligible) unclassified Methanobacterium genus (Euryarchaeota phylum). Interestingly, the reduction of relative abundance of MA was 39.17%, very close to the percent reduction of CH4 production, 38%. Our data showed that there was a parallel and similar percent decrease of both CH4 production and relative abundance of the predominant MA in our experiment, although the statistical significance was not complete. Finally, our results hold promise for significantly decreasing ruminal CH4 by 38%. Thus, our work is one step toward the sustainable management of the livestock sector.

scholarly journals Lovastatin as a supplement to mitigate rumen methanogenesis: an overview

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Amaury Ábrego-Gacía ◽  
Héctor M. Poggi-Varaldo ◽  
Vania Robles-González ◽  
Teresa Ponce-Noyola ◽  
Graciano Calva-Calva ◽  
...  
Keyword(s):  
Volatile Fatty Acids ◽  
Methanogenic Archaea ◽  
Methane Emissions ◽  
Ruminal Fermentation ◽  
Enteric Fermentation ◽  
Feed Composition ◽  
Rumen Microbiota ◽  
Alpha And Beta Diversity

AbstractMethane from enteric fermentation is the gas with the greatest environmental impact emitted by ruminants. Lovastatin (Lv) addition to feedstocks could be a strategy to mitigate rumen methane emissions via decreasing the population of methanogenic archaea (MA). Thus, this paper provides the first overview of the effects of Lv supplementation, focusing on the inhibition of methane production, rumen microbiota, and ruminal fermentation. Results indicated that Lv treatment had a strong anti-methanogenic effect on pure strains of MA. However, there are uncertainties from in vitro rumen fermentation trials with complex substrates and rumen inoculum.Solid-state fermentation (SSF) has emerged as a cost-effective option to produce Lv. In this way, SSF of agricultural residues as an Lv-carrier supplement in sheep and goats demonstrated a consistent decrease in ruminal methane emissions. The experimental evidence for in vitro conditions showed that Lv did not affect the volatile fatty acids (VFA). However, in vivo experiments demonstrated that the production of VFA was decreased. Lv did not negatively affect the digestibility of dry matter during in vitro and in vivo methods, and there is even evidence that it can induce an increase in digestibility. Regarding the rumen microbiota, populations of MA were reduced, and no differences were detected in alpha and beta diversity associated with Lv treatment. However, some changes in the relative abundance of the microbiota were induced. Further studies are recommended on: (i) Lv biodegradation products and stability, as well as its adsorption onto the solid matter in the rumen, to gain more insight on the “available” or effective Lv concentration; and (ii) to determine whether the effect of Lv on ruminal fermentation also depends on the feed composition and different ruminants.

Effect of pine-based biochars with differing physiochemical properties on methane production, ruminal fermentation, and rumen microbiota in an artificial rumen (RUSITEC) fed barley silage

2021 ◽  
pp. 1-13
Author(s):  
Paul Tamayao ◽  
Gabriel O. Ribeiro ◽  
Tim A. McAllister ◽  
Kim H. Ominski ◽  
Atef M. Saleem ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Fixed Effects ◽  
Block Design ◽  
Rumen Fermentation ◽  
Ruminal Fermentation ◽  
Microbial Protein ◽  
Rumen Microbiota ◽  
Alpha And Beta Diversity ◽  
Microbial Protein Synthesis ◽  
Barley Silage

This study investigated the effects of three pine-based biochar products on nutrient disappearance, total gas and methane (CH4) production, rumen fermentation, microbial protein synthesis, and rumen microbiota in a rumen simulation technique (RUSITEC) fed a barley-silage-based total mixed ration (TMR). Treatments consisted of 10 g TMR supplemented with no biochar (control) and three different biochars (CP016, CP024, and CP028) included at 20 g·kg−1 DM. Treatments were assigned to 16 fermenters (n = 4 per treatment) in two RUSITEC units in a randomized block design for a 17 d experimental period. Data were analyzed using MIXED procedure in SAS, with treatment and day of sampling as fixed effects and RUSITEC unit and fermenters as random effects. Biochar did not affect nutrient disappearance (P > 0.05), nor total gas or CH4, irrespective of unit of expression. The volatile fatty acid, NH3-N, total protozoa, and microbial protein synthesis were not affected by biochar inclusion (P > 0.05). Alpha and beta diversity and rumen microbiota families were not affected by biochar inclusion (P > 0.05). In conclusion, biochar did not reduce CH4 emissions nor affect nutrient disappearance, rumen fermentation, microbial protein synthesis, or rumen microbiota in the RUSITEC.

BH3 Profiling As Predictor Of 5-Azacytidine and Decitabine Clinical Responses

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 603-603 ◽  
Author(s):  
Raoul Tibes ◽  
Steven M. Kornblau ◽  
William E. Pierceall ◽  
Ryan Lena ◽  
Yi Hua Qiu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Statistical Significance ◽  
Family Members ◽  
Specific Inhibitor ◽  
Hypomethylating Agents ◽  
Employment Equity ◽  
Number Of Patients ◽  
Clinical Responses ◽  
Equity Ownership

Abstract Hypomethylating agents have changed treatment and outcomes in MDS and are active in AML patients. However a significant number of patients do not respond to 5-Azacytidine or Decitabine and there is no clinically validated assay that predicts response to 5-Azacytidine (5-Aza) or Decitabine (DAC). Clinical parameters and recently TET2 mutations have been identified as genetic predictors of response to 5-Aza. Several recent papers report on the possible predictive value of anti-apoptotic proteins, for example BCL2L10. In previous genome-scale RNA-interference (RNAi) screens anti-apoptotic BCL-2 family members, most potently BCL-XL, were identified as top targets whose down-regulation sensitize to 5-Aza. Results were confirmed with the BCL-XL and BCL-2 inhibitor ABT-737, as well as the BCL-2-specific inhibitor ABT-199, both of which sensitized myeloid cells to 5-Aza. Although it is expected that interfering with anti-apoptotic genes would sensitize to hypomethylating similar to cytotoxics, few of the other >900 genes from our RNAi sensitizer screens, including few of the 571 kinases, sensitized to 5-Aza as potently as inhibition of BCL-XL. Thus BCL-2 family members constitute an especially potent context to modulate the activity of 5-Aza. To assess if protein expression of the top targets BCL-XL, BCL-2 or MCL-1 correlate with and may explain a preferential sensitization, 577 primary AML samples were examined by Reverse Phase Protein Array (RPPA). There was substantial overlap of expression across and within FAB subgroups and cytogenetics, and expression of neither of these genes/proteins could explain the observed effects. This may be expected due to functional redundancies amongst the different anti-apoptotic BCL-2 family members, as well as distinct, yet overlapping binding affinities between anti- and pro-apoptotic BCL-2 proteins and BH3 peptides. Thus, we next aimed to functionally interrogate the overall balance of pro- and anti-apoptotic BCL-2 family members, also described as “primedness” by BH3 profiling. This assay uses peptides derived from pro-apoptotic BH3-only proteins to determine the capacity of cells to initiate apoptosis in response to pro-apoptotic BH3 molecules [anti-apoptotic BCL-2 molecules like BCL-XL and BCL-2 bind and inhibit BH3-only molecules]. This assay is a broad functional readout that incorporates many parameters including the consequences of varying BCL2L10 expression. To establish a proof-of-concept that the functional interrogation of specific apoptotic thresholds may be an optimal readout for 5-Aza response, we first assessed a broad panel of 13 AML-derived cell lines by BH3 profiling and in parallel 5-Aza drug dose response experiments. Several BH3 profiling metrics, including NOXA plus BIM, significantly correlated with 5-Aza sensitivity in vitro (Figure 1). Next, to correlate actual clinical responses to 5-Aza with BH3 profiling, specimens from 28 AML, MDS and MDS/MPN overlap patients treated with 5-Aza or DAC-based regimens and for whom clinical outcome was available, were assayed in BH3 assays. In the clinic, the best BH3 metrics were combined values from NOXA and BIM peptides similar to cell lines. NOXA plus BIM discriminated clinical responses defined as achieving any response (sensitive) versus a patient being resistance/refractory to 5-Aza/DAC based regimens with statistical significance (Mann-Whitney two-tailed p = 0.001). This was true for the entire group of patients (5-Aza- and DAC- regimens) with a Receiver Operating Characteristic (ROC) of an AUC=0.875, with a further increase for patients treated only with 5-Aza based regimens with an AUC=0.95.Figure 1BH3 metrics distinguish cell lines with higher EC 50 (> 2uM) from those with lower EC50 (generally < 1uM).Figure 1. BH3 metrics distinguish cell lines with higher EC 50 (> 2uM) from those with lower EC50 (generally < 1uM). In conclusion, due to significant expression overlap and functional redundancies of the BCL-2 family proteins, expression does not correlate with 5-Aza clinical or in vitro responses. However, BH3 profiling as a general functional readout of apoptotic primedness, significantly discriminated responses in patients as well as in vitro. Thus, BH3 profiling incorporates the entirety of pro- and anti-apoptotic molecules (including BCL2L10) and is promising to predict responses to hypomethylating agents. We propose to prospectively validate BH3 profiling as a predictive biomarker assay for 5-Aza or DAC based regimens. Disclosures: Pierceall: Eutropics: Employment, Equity Ownership. Lena:Eutropics: Employment, Equity Ownership. Cardone:Eutropics: Employment, Equity Ownership. Elashoff:Eutropics: Employment. Blake:Noel Blake: Employment, Equity Ownership.

scholarly journals Roughage to Concentrate Ratio and Saccharomyces cerevisiae Inclusion Could Modulate Feed Digestion and In Vitro Ruminal Fermentation

2020 ◽  
Vol 7 (4) ◽  
pp. 151
Author(s):  
Kampanat Phesatcha ◽  
Burarat Phesatcha ◽  
Metha Wanapat ◽  
Anusorn Cherdthong
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Ratio ◽  
Gas Production ◽  
Control Treatment ◽  
Ruminal Fermentation ◽  
Dry Matter Digestibility ◽  
Ch4 Production ◽  
Feed Digestibility ◽  
Live Yeast

The objective of this research was to investigate the effect of the roughage-to-concentrate (R:C) ratio and the addition of live yeast (LY) on ruminal fermentation characteristics and methane (CH4) production. The experimental design was randomly allocated according to a completely randomized design in a 4 × 4 factorial arrangement. The first factor was four rations of R:C at 80:20, 60:40, 40:60, and 20:80, and the second factor was an additional four doses of Saccharomyces cerevisiae (live yeast; LY) at 0, 2.0 × 106, 4.0 × 106, and 6.0 × 106 colony-forming unit (cfu), respectively. For the in vitro method, during the incubation, the gas production was noted at 0, 1, 2, 4, 6, 8, 10, 12, 18, 24, 48, 72, and 96 h. The rumen solution mixture was collected at 0, 4, 8, 12, and 24 h of incubating after inoculation. Cumulative gas production at 96 h was highest in the R:C ratio, at 20:80, while the addition of LY improves the kinetics and accumulation of gas (p > 0.05). Maximum in vitro dry matter digestibility (IVDMD) and in vitro organic matter digestibility (IVOMD) at 24 h after incubation were achieved at the R:C ratio 20:80 and the addition of LY at 6 × 106 cfu, which were greater than the control by 13.7% and 12.4%, respectively. Ruminal pH at 8 h after incubation decreased with an increased proportion of concentrates in the diet, whereas it was lowest when the R:C ratio was at 20:80. Increasing the proportion of a concentrate diet increased total volatile fatty acid (TVFA) and propionic acid (C3), whereas the acetic acid (C2) and C2-to-C3 ratios decreased (p < 0.05). TVFA and C3 increased with the addition of LY at 6 × 106 cfu, which was greater than the control by 11.5% and 17.2%, respectively. No interaction effect was observed between the R:C ratio and LY on the CH4 concentration. The calculated ruminal CH4 production decreased with the increasing proportion of concentrates in the diet, particularly the R:C ratio at 20:80. The CH4 production for LY addition at 6 × 106 cfu was lower than the control treatment by 17.2%. Moreover, the greatest populations of bacteria, protozoa, and fungi at 8 h after incubation were found with the addition of LY at 6 × 106 cfu, which were higher than the control by 19.0%, 20.7%, and 40.4%, respectively. In conclusion, a high ratio of roughage and the concentrate and addition of LY at 6.0 × 106 cfu of the total dietary substrate could improve rumen fermentation, improve feed digestibility, and reduce the CH4 production.

Preliminary study on the effects of ammonium nicotinate on in vitro ruminal fermentation as determined using rumen simulation technique (Rusitec)

2011 ◽  
Vol 51 (3) ◽  
pp. 233 ◽  
Author(s):  
Carla R. Soliva ◽  
Carmen Kunz
Keyword(s):  
In Vitro Study ◽  
Basal Diet ◽  
Short Chain Fatty Acids ◽  
Simulation Technique ◽  
Control Treatment ◽  
Lower Amount ◽  
Ruminal Fermentation ◽  
The Third ◽  
Low Protein

The objective of the present in vitro study was to investigate the effects of different dietary supplementation levels of ammonium nicotinate (NA-NH4), a precursor product when manufacturing nicotinic acid (NA), on ruminal fermentation traits. Four experimental runs were carried out incubating ruminal fluid from a donor cow by using rumen simulation technique (Rusitec). A low-protein (109 g/kg feed dry matter) basal diet, consisting of maize silage, hay and concentrate, served as the first control. Supplements were NA at 4.7 mg/day (second control), NA-NH4 at supplementation rates of 2.7, 5.4, 10.7 and 21.4 mg/day, or NH4-sulfate at 2.53 mg/day (the third control). All NA-containing treatments were supplemented with the same amount of sulfate as supplied with the third control treatment. None of the NA-supplements affected any of the fermentation traits significantly compared with the first control treatment, except for a decrease in total short-chain fatty acids at the highest supplementation rate of NA-NH4. No differences were found between the treatments containing the same amount of NA, i.e. the second control and the NA-NH4 treatment at 5.4 mg/day. Comparing the different NA-NH4 supplementation rates revealed that 5.4 mg/day of NA-NH4 resulted in a lower amount of nitrogen (N) recovered in ammonia than the highest NA-NH4 supplementation rate, and increased non-ammonia N. The findings confirmed the suitability of NA-NH4, instead of pure NA, as a feed supplement; however, increasing NA-NH4-supplementation above the typical rate for dairy cattle might negatively affect ruminal fermentation.

scholarly journals Effects of Different Combinations of Sugar and Starch Concentrations on Ruminal Fermentation and Bacterial-Community Composition in vitro

2021 ◽  
Vol 8 ◽  
Author(s):  
Jia-nan Dong ◽  
Song-ze Li ◽  
Xue Chen ◽  
Gui-xin Qin ◽  
Tao Wang ◽  
...  
Keyword(s):  
Bacterial Community ◽  
Production Rate ◽  
Community Composition ◽  
Relative Abundance ◽  
Dry Matter ◽  
Bacterial Community Composition ◽  
Rumen Fermentation ◽  
Gas Production ◽  
Ruminal Fermentation

High levels of starch is known to have positive effects on both energy supply and milk yield but increases the risk of rumen acidosis. The use of sugar as a non-structural carbohydrate could circumvent this risk while maintaining the benefits, but its effects and that of the simultaneous use of both sugar and starch are not as well-understood. This study aimed to evaluate the effects of different combinations of sugar and starch concentrations on ruminal fermentation and bacterial community composition in vitro in a 4 ×4 factorial experiment. Sixteen dietary treatments were formulated with 4 levels of sugar (6, 8, 10, and 12% of dietary dry matter), and 4 levels of starch (21, 23, 25, and 27% of dietary dry matter). Samples were taken at 0.5, 1, 3, 6, 12, and 24 h after cultivation to determine the disappearance rate of dry matter, rumen fermentation parameters and bacterial community composition. Butyric acid, gas production, and Treponema abundance were significantly influenced by the sugar level. The pH, acetic acid, and propionic acid levels were significantly influenced by starch levels. However, the interactive effect of sugar and starch was only observed on the rate of dry matter disappearance. Furthermore, different combinations of starch and sugar had different effects on volatile fatty acid production rate, gas production rate, and dry matter disappearance rate. The production rate of rumen fermentation parameters in the high sugar group was higher. Additionally, increasing the sugar content in the diet did not change the main phylum composition in the rumen, but significantly increased the relative abundance of Bacteroidetes and Firmicutes phyla, while the relative abundance of Proteobacteria was reduced. At the genus level, the high glucose group showed significantly higher relative abundance of Treponema (P &lt; 0.05) and significantly lower relative abundance of Ruminobacter, Ruminococcus, and Streptococcus (P &lt; 0.05). In conclusion, different combinations of sugar and starch concentrations have inconsistent effects on rumen fermentation characteristics, suggesting that the starch in diets cannot be simply replaced with sugar; the combined effects of sugar and starch should be considered to improve the feed utilization rate.

scholarly journals EXTRATO DE MAMONA COMO MANIPULADOR DA FERMENTAÇÃO RUMINAL

Nativa ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 289
Author(s):  
Pâmella Moraes Franco ◽  
Márcia Rodrigues Carvalho Oliveira ◽  
Joao Rafael de Assis ◽  
Jurandy Gouveira Junior ◽  
Rodrigo Nazare Santos Torres ◽  
...  
Keyword(s):  
Gas Production ◽  
Control Treatment ◽  
Energetic Efficiency ◽  
Ruminal Fermentation ◽  
Ruminal Ph ◽  
Dietary Condition ◽  
Randomized Design ◽  
Completely Randomized Design ◽  
High Forage

Objetivou-se investigar os efeitos da adição do extrato de farelo de mamona (EFM) sobre o perfil da fermentação ruminal in vitro em dietas com alto e baixo teor de forragem. Utilizou-se ensaio de incubação ruminal in vitro com dois controles, um negativo (sem aditivo) e um positivo (monensina sódica) e EFM liofilizado (20, 40 e 60 mg/frasco). Em condições de alto teor de forragem na dieta, a adição do EFM aumentou o pH do meio e a concentração de acetato, reduziu a produção de gás, mas não afetou a produção de gás por unidade de matéria seca (MS) digerida em relação ao tratamento controle. Em comparação com monensina sódica, o EFM reduziu as concentrações de propionato e amônia e aumentou a produção de gás por unidade de MS digerida. Em condições de baixo teor de forragem, a adição do EFM reduziu o pH e potencial redox do meio em relação ao tratamento controle. Em comparação com a monensina sódica, o EFM reduziu o pH do meio e a produção total de gás, mas não afetou a produção de gás por unidade de MS digerida. O extrato de farelo de mamona destoxificado não apresenta potencial como manipulador da fermentação ruminal.Palavras-chave: amônia; digestibilidade; eficiência; metano. CASTOR BEAN EXTRACT AS A MANIPULATOR OF RUMINAL FERMENTATION ABSTRACT: Effects of the castorbean meal extract (CME) on ruminal in vitro were investigate in high and low forage diet conditions. For each dietary condition, one in vitro ruminal incubation experiment was conducted in a completely randomized design, with nine repetitions per treatment (three animal inoculum donators and three 48 hors-incubations). In high forage diet, CME increased ruminal pH acetate concentration, reduced gas production, but it did not affect the gas production per unit of digested dry matter (DM), in relation to control treatment. Compare to monensin sodium, CME reduced propionate and ammonia concentrations and increased gas production per unit of digested DM, indicating that CME reduces ruminal energetic efficiency. In low forage diet, CME reduced pH and redox potential compare to control. Compare to monensin sodium, CME reduced pH and gas production, but it did not affect gas production per unit of digested DM. Castorbean meal extract does not present potential as manipulator of the ruminal fermentation.Keywords: ammonia; digestibility; efficiency; methane.

scholarly journals Impacts of Mootral on Methane Production, Rumen Fermentation, and Microbial Community in an in vitro Study

2021 ◽  
Vol 7 ◽  
Author(s):  
Eslam Ahmed ◽  
Rintaro Yano ◽  
Miho Fujimori ◽  
Deepashree Kand ◽  
Masaaki Hanada ◽  
...  
Keyword(s):  
Microbial Community ◽  
Relative Abundance ◽  
Methane Production ◽  
Statistical Significance ◽  
Archaeal Community ◽  
Rumen Fermentation ◽  
Mitigation Strategies ◽  
Dependent Manner ◽  
Dose Dependent

Methane mitigation strategies have a two-sided benefit for both environment and efficient livestock production. This preliminary short-term in vitro trial using Mootral (garlic and citrus extracts), a novel natural feed supplement, was conducted to evaluate its efficacy on rumen fermentation characteristics, methane production, and the bacterial and archaeal community. The experiment was performed as a batch culture using rumen fluid collected from sheep, and Mootral was supplemented in three concentrations: 0% (Control), 10%, and 20% of the substrate (50% Grass:50% Concentrate). The rumen fermentation data and alpha diversity of microbial community were analyzed by ordinary one-way analysis of variance. The relative abundance and statistical significance of families and operational taxonomic units (OTUs) among the groups were compared by Kruskal–Wallis H test using Calypso software. After 24-h incubation at 39°C, Mootral in a dose-dependent manner improved the production of total volatile fatty acids and propionate while it reduced the acetate proportion and acetate/propionate ratio. The total produced gas was two times higher in the Mootral-supplemented groups than control (P &lt; 0.01), while the proportion of methane in the produced gas was reduced by 22% (P &lt; 0.05) and 54% (P &lt; 0.01) for 10 and 20% Mootral, respectively. Mootral did not change pH, digestibility, and ammonia-nitrogen. Microbial community analyses showed that Mootral effectively changed the ruminal microbiome. The bacterial community showed an increase of the relative abundance of the propionate-producing family such as Prevotellaceae (P = 0.014) and Veillonellaceae (P = 0.030), while there was a decrease in the relative abundance of some hydrogen-producing bacteria by Mootral supplementation. In the archaeal community, Methanobacteriaceae was decreased by Mootral supplementation compared with control (P = 0.032), while the Methanomassiliicoccaceae family increased in a dose-dependent effect (P = 0.038). The results of the study showed the efficacy of the new mixture to alter the ruminal microbial community, produce more propionate, and reduce microbial groups associated with methane production, thus suggesting that Mootral is a promising natural mixture for methane reduction from ruminants.

scholarly journals Óleos essenciais como modificadores da fermentação ruminal em substituição a monensina sódica in vitro

2019 ◽  
Vol 68 (264) ◽  
pp. 576-581
Author(s):  
L. Antunes Stella ◽  
V. Rosa Prates ◽  
A. Zubieta ◽  
C. Bayer ◽  
J.O. Jardim Barcellos
Keyword(s):  
Gas Production ◽  
Control Treatment ◽  
Clove Oil ◽  
Ruminal Fermentation ◽  
Cinnamon Oil ◽  
Bitter Orange ◽  
Randomized Design ◽  
Completely Randomized Design ◽  
Fermentation Parameters

The objective of this study to evaluate the effect of secondary plant compounds present in essential oils in replacement of monensin on in vitro ruminal fermentation parameters. It was adopted a completely randomized design with nine treatments and four replicates. The treatments were: control (CON), monensin (MON), garlic oil (ALH), cinnamon oil (CAN), clove oil (CRA), mint oil (HOR), juniper oil (JUN), bitter orange oil (LAR), and melaleuca oil (MEL). The in vitro gas technique was used to record total gas production at 4, 8, 12 and 24 h after incubation. MON, CAN and CRA increased gas production Only the garlic and cinnamon treatments reduced the digestibility of organic matter in 20 and 26% in relation to the control treatment. Methane production reduced (P

scholarly journals 237EFFECTS OF IN VITRO V. IN VIVO CULTURE ON EXPRESSION OF EMBRYO DERIVED GENE TRANSCRIPTS INVOLVED IN APOPTOSIS IN SINGLE BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 239
Author(s):  
H.M. Knijn ◽  
C. Wrenzycki ◽  
P.L.A.M. Vos ◽  
G.C. van der Weijden ◽  
H. Niemann ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Relative Abundance ◽  
Molecular Basis ◽  
Culture Medium ◽  
Specific Inhibitor ◽  
Control Group ◽  
Cell Stage ◽  
Gene Transcripts

Earlier studies reported that the level of apoptosis in in vitro-produced bovine blastocysts is higher then in in vivo developed blastocysts (Gjorret et al., 2003: Biol. Reprod, in press). The molecular basis for this difference has not yet been studied. The regulation and execution of apoptosis is dependent on a cascade of events in which many proteins are involved. The aim of the present study was to analyze expression of BAX, a pro-apoptotic, and BCL-XL, a anti-apoptotic member of the BCL-2 family, and heat shock protein (HSP 70.1) transcripts in blastocysts cultured in vitro or in vivo. Furthermore, to verify if these transcripts detected in the blastocysts stages were newly expressed from the embryonic genome, a RNA polymerase II specific inhibitor, α-amanitin, was added to the culture medium from the zygote stage onwards, to block transcription. For the in vitro group, embryos were obtained from abattoir oocytes after IVM/IVF and IVC in SOF medium. For the in vivo group, embryos were collected from normally cyclic cows, superovulated with 3000 IU eCG (Intergonan;; Intervet, Tönisvorst, Germany) at day 7 po by non-surgical uterine flushing. The developmental stage of the embryos was determined with stereomicroscopy, and early blastocysts (eb), blastocysts (b), and expanded blastocysts (xb) were collected and frozen at −80°C. For transcription inhibition experiment, embryos were cultured in vitro as for the in vitro group until the 8- to 16-cell stage at 100h after the start of fertilization, one group with 10mM α-amanitin added to the culture medium (α-amanitin group) and the other group without (control group). A highly sensitive semi-quantitative RT-PCR assay (Wrenzycki et al., 1999) Mol. Reprod. Dev. 53, 8–18) was used to determine the relative levels of gene transcripts in single blastocysts and pooled 8- to 16-cell embryos. The relative abundance was calculated on a per cell basis per embryo. Assays were repeated on average eight times. Data on the relative expression of transcripts in blastocysts were analyzed by Anova followed by multiple pairwise comparisons using the Tukey test. No significant differences in relative abundance between in vitro and in vivo cultured embryos, for any of the developmental stages, were found for the three apoptosis related genes. The molecular basis for the difference in level of apoptosis between in vitro and in vivo cultured blastocysts is not related to the level of expression of BAX, BCL-XL and HSP transcripts but other genes involved in the apoptotic cascade may be responsible for the reported differences. The expression of BCL-XL and HSP 70.1 transcripts in the blastocysts was from embryonic origin as no expression of these transcripts was detected in the 8- to 16-cell stage embryos treated with α-amanitin. The expression of BAX gene transcripts was not affected by α-amanitin. Probably the maternally derived transcripts are very stable and not yet degraded at the 8- to 16-cell stage. Table 1 Relative abundance (±SEM) of BAX, BCL-XL, and HSP 70.1 transcripts in bovine single in vitro or in vivo-produced eb, b, and xb and in vitro produced pooled 8- to 16-cell stage embryos treated with or without α-amanitin

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