Short communication: Short-term effect of 3-nitrooxypropanol on feed dry matter intake in lactating dairy cows.

The objective of this study was to investigate the effect of 3-nitrooxypropanol (3-NOP), an enteric methane inhibitor under investigation, on short-term dry matter intake (DMI) in lactating dairy cows. Following a 1-wk adaptation period, 12 multiparous Holstein cows were fed a basal total mixed ration (TMR) containing increasing levels of 3-NOP during 5 consecutive, 6-d periods. The experiment was conducted in a tiestall barn. Feed bins were split in half by a solid divider, and cows simultaneously received the basal TMR supplemented with the following: (1) a placebo without 3-NOP or (2) 3-NOP included in the TMR at 30, 60, 90, or 120 mg/kg of feed dry matter (experimental periods 2, 3, 4, and 5, respectively). Cows received the control diet (basal TMR plus placebo premix) during experimental period 1. A premix containing ground corn grain, soybean oil, and dry molasses was used to incorporate 3-NOP in the ration. Cows were fed twice daily as follows: 60% of the daily feed allowance at 0800 h and 40% at 1800 h. Feed offered and refused was recorded at each feeding. During the morning feedings, each cow was offered either control or 3-NOP-treated TMR at 150% of her average intake during the previous 3 d. After collection of the evening refusals, cows received only the basal TMR without the premix until the next morning feeding. The test period for the short-term DMI data collection was defined from morning feeding to afternoon refusals collection during each day of each experimental period. Location (left or right) of the control and 3-NOP diets within a feed bin was switched every day during each period to avoid feed location bias. Dry matter intake of TMR during the test period was quadratically increased by 3-NOP compared with the control. Inclusion of 3-NOP at 120 mg/kg of feed dry matter resulted in decreased 10-h DMI compared with the lower 3-NOP doses, but was similar to the control. There was no effect of feed location (left or right) within feed bin on DMI. Data from this short-term study suggests that 3-NOP does not have a negative effect on DMI in lactating dairy cows.

Potential roles of nitrate and live yeast culture in suppressing methane emission and influencing ruminal fermentation, digestibility, and milk production in lactating Jersey cows.

Concern over the carbon footprint of the dairy industry has led to various dietary approaches to mitigate enteric CH4 production. One approach is feeding the electron acceptor NO3-, thus outcompeting methanogens for aqueous H2. We hypothesized that a live yeast culture (LYC; Saccharomyces cerevisiae from Yea-Sacc 1026, Alltech Inc., Nicholasville, KY) would stimulate the complete reduction of NO3- to NH3 by selenomonads, thus decreasing the quantity of CH4 emissions per unit of energy-corrected milk production while decreasing blood methemoglobin concentration resulting from the absorbed intermediate, NO2-. Twelve lactating Jersey cows (8 multiparous and noncannulated; 4 primiparous and ruminally cannulated) were used in a replicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments. Cattle were fed diets containing 1.5% NO3- (from calcium ammonium nitrate) or an isonitrogenous control diet (containing additional urea) and given a top-dress of ground corn without or with LYC, with the fourth week used for data collection. Noncannulated cows were spot measured for CH4 emission by mouth using GreenFeed (C-Lock Inc., Rapid City, SD). The main effect of NO3- decreased CH4 by 17% but decreased dry matter intake by 10% (from 19.8 to 17.8 kg/d) such that CH4:dry matter intake numerically decreased by 8% and CH4:milk net energy for lactation production was unaffected by treatment. Milk and milk fat production were not affected, but NO3- decreased milk protein from 758 to 689 g/d. Ruminal pH decreased more sharply after feeding for cows fed diets without NO3-. Acetate:propionate was greater for cows fed NO3-, particularly when combined with LYC (interaction effect). Blood methemoglobin was higher for cattle fed NO3- than for those fed the control diet but was low for both treatments (1.5 vs. 0.5%, respectively; only one measurement exceeded 5%), indicating minimal risk for NO2- accumulation at our feeding level of NO3-. Although neither apparent organic matter nor neutral detergent fiber digestibilities were affected, apparent N digestibility had an interaction for NO3- × LYC such that apparent N digestibility was numerically lowest for diets containing both NO3- and LYC compared with the other 3 diets. Under the conditions of this study, NO3- mitigated ruminal methanogenesis but also depressed dry matter intake and milk protein yield. Based on the fact that few interactions were detected, LYC had a minimal role in attenuating negative cow responses to NO3- supplementation.

Rapid Communication: Evaluation of methane inhibitor 3-nitrooxypropanol and monensin in a high-grain diet using the rumen simulation technique (Rusitec).

The objective of this study was to evaluate the effects of 3-nitrooxypropanol (NOP), a known methane (CH) inhibitor; the ionophore monensin (MON); and their combination on in vitro CH production in a high-grain diet (85% barley grain, 10% barley silage, and 5% vitamin-mineral supplement; DM basis) using a rumen simulation technique (Rusitec). Sixteen fermentation vessels in 2 Rusitec apparatuses (blocks) were used in a completely randomized block design with 4 treatments: Control, NOP (200 µg/g DM), MON (200 µg/g DM), and the combination of 200 µg NOP/g DM and 200 µg MON/g DM (NOP + MON). Two fermenters within each apparatus were randomly assigned to a treatment. Treatments were mixed with 10 g of substrate and supplied on a daily basis. The study included an 8-d adaptation period without treatment supplementation and a 6-d period for addition of treatments. Dry matter disappearance, pH, and total VFA were not affected by treatment ( ≥ 0.34). Acetate proportion was decreased by 8.3% and 14.9% with NOP and NOP + MON ( < 0.01), respectively; however, propionate proportion was not affected by treatment ( = 0.44). The acetate to propionate ratio was lowered by 21.1% with the combination of NOP and MON ( = 0.02), whereas ammonia-N concentration was not affected by treatment ( = 0.50). Total gas production was unaffected ( = 0.50), but CH production decreased by 77.7% and 75.95% ( < 0.01) with NOP and NOP + MON addition, respectively. Concurrently, H gas production increased by 131.3% and 185.6% ( = 0.01) with NOP and NOP + MON treatments, respectively. The copy number of methanogens was decreased in both solid and liquid phases ( < 0.01) with NOP and NOP + MON treatments. Despite the combination of NOP + MON showing the greatest decrease in acetate molar proportion and acetate to propionate ratio, it did not further inhibit CH beyond the effect of NOP alone. The decrease in CH emissions with treatments that included NOP occurred along with a decrease in the copy number of methanogens associated with the solid and liquid phases, confirming the inhibitory effects of NOP on these microorganisms. In conclusion, the combined effects of NOP and MON on CH mitigation did not exceed the effect of NOP alone when using a high-grain diet in vitro.

Bioactive fractions from the pasture legume Biserrula pelecinus L. have an anti-methanogenic effect against key rumen methanogens.

Methanogenic archaea (methanogens) are common inhabitants of the mammalian intestinal tract. In ruminants, they are responsible for producing abundant amounts of methane during digestion of food, but selected bioactive plants and compounds may inhibit this activity. Recently, we have identified that, Biserrula pelecinus L. (biserrula) is one such plant and the current study investigated the specific anti-methanogenic activity of the plant. Bioassay-guided extraction and fractionation, coupled with in vitro fermentation batch culture were used to select the most bioactive fractions of biserrula. The four fractions were then tested against five species of methanogens grown in pure culture. Fraction bioactivity was assessed by measuring methane production and amplification of the methanogen mcrA gene. Treatments that showed bioactivity were subcultured in fresh broth without the bioactive fraction to distinguish between static and cidal effects. All four fractions were active against pure cultures, but the F2 fraction was the most consistent inhibitor of both methane production and cell growth, affecting four species of methanogens and also producing equivocal-cidal effects on the methanogens. Other fractions had selective activity affecting only some methanogens, or reducing either methane production or methanogenic cell growth. In conclusion, the anti-methanogenic activity of biserrula can be linked to compounds contained in selected bioactive fractions, with the F2 fraction strongly affecting key rumen methanogens. Further study is required to identify the specific plant compounds in biserrula that are responsible for the anti-methanogenic activity. These findings will help devise novel strategies to control methanogen populations and activity in the rumen, and consequently contribute in reducing greenhouse gas emissions from ruminants.

Effects of essential oils from medicinal plants acclimated to Benin on in vitro ruminal fermentation of Andropogon gayanus grass.

BACKGROUND: Plants from West Africa commonly used in both human and veterinary medicine contain various secondary metabolites. However, their potential in mitigating ruminal methane production has not been explored. This study examined the effects of seven essential oils (EOs) from plants acclimated to Benin at four dosages (100, 200, 300 and 400 mg L(-1)), on in vitro rumen microbial fermentation and methane production using Andropogon gayanus grass as a substrate. RESULTS: Compared to control, Laurus nobilis (300-400 mg L(-1) ), Citrus aurantifolia (300-400 mg L(-1)) and Ocimum gratissimum (200-400 mg L(-1)) decreased (P < 0.05) methane production (mL g(-1) DM) by 8.1-11.8%, 11.9-17.8% and 7.9-30.6%, respectively. Relative to the control, reductions in methane (mL g(-1) DM) of 11.4%, 13.5% and 14.2% were only observed at 400 mg L(-1) for Eucalyptus citriodora, Ocimum basilicum and Cymbopogon citratus, respectively. These EOs lowered methane without reducing concentrations of total volatile fatty acids or causing a shift from acetate to propionate production. All EOs (except M. piperita) reduced (P < 0.05) apparent dry matter (DM) disappearance of A. gayanus. CONCLUSIONS: The current study demonstrated that EOs from plants grown in Benin inhibited in vitro methane production mainly through a reduction in apparent DM digestibility.

Assessment of the effect of condensed (acacia and quebracho) and hydrolysable (chestnut and valonea) tannins on rumen fermentation and methane production in vitro.

BACKGROUND: Tannins added to animal diets may have a positive effect on energy and protein utilisation in the rumen. The objective of this study was to examine the impact of different sources and concentrations (20, 50, 100, 150 and 200 g kg⁻¹ dry matter (DM)) of condensed (acacia and quebracho) and hydrolysable (chestnut and valonea) tannins on rumen microbial fermentation in vitro. The experiment also included a negative control with no tannins (control) and a positive control with monensin (10 mg L⁻¹). RESULTS: In vitro gas production and total volatile fatty acid (VFA) concentration decreased as tannin concentration increased. Addition of acacia, chestnut or valonea tannins at ≥ 50 g kg⁻¹ or quebracho tannins at ≥ 100 g kg⁻¹ resulted in a decrease (up to 40%) in methane (CH4) production compared with the control. Valonea tannins were the only tannin source that reduced (-11%) CH4 production at 50 g kg⁻¹ without affecting VFA concentration. Tannin treatments reduced ammonia (NH3) and branched-chain VFA concentrations, indicating a reduction in ruminal protein degradation. Monensin reduced CH4 production (-37%) and NH3 concentration (-20%) without affecting total VFA concentration. CONCLUSION: Supplying acacia, chestnut or valonea tannins at 50 g kg⁻¹ has the potential to reduce CH4 production and ruminal protein degradation with minimum detrimental effects on efficiency of ruminal fermentation.

Effect of methane inhibitors on the metabolism of rumen microbes in vitro.

In incubations in vitro with rumen fluid, the effect of two methane inhibitors, linseed oil hydrolysate (LOH) and chloral hydrate (CH) on the efficiency of microbiol growth was investigated. Total and net microbial growth were determined from 32PO43- and NH3--N incorporation respectively and expressed as g N incorporated per kg organic matter fermented (gN/kgOMf). In a first series on incubations, it was found that LOH had no influence on overall microbial growth efficiency, while with CH, a small but significant decrease of total and net growth efficiency was measured. Further experiments showed that this was not due to accumulation of hydrogen gas in the CH incubations. Microscopic examination showed a toxic effect of LOH on protozoa, but with CH, no such effect was observed. This observation, together with earlier work where a considerable increase in microbial growth efficiency was found in vitro after defaunation of the rumen suggested the following hypothesis: both inhibitors lowered bacterial growth. In the case of LOH, this effect is marked by the defaunating action of LOH, the latter resulting in an increased growth efficiency of the bacterial fraction. This hypothesis was confirmed by incubations with washed cell suspensions (WCS) of mixed rumen bacteria, where growth efficiency was indeed decreased by both inhibitors. The possible mechanism explaining this phenomenon was discussed.