Anne Louise Frydendahl Hellwing

Beets have a high sugar content, which is primarily fermented to ethanol during ensiling. This study investigated the effects of feeding diets with sugar-rich beets or ethanol-rich beet silage to lactating dairy cows on postabsorptive ethanol metabolism, enteric gas emissions, and microbial protein synthesis. Beets were washed, crushed, and stored in daily portions either as is (frozen at -20°C for preservation) or ensiled in barrels. Four rumen-cannulated Danish Holstein cows were used in a replicated (one complete and one incomplete) 3 × 3 Latin square design, with 3 treatments and three 21-d periods. The control diet (CON) consisted of grass-clover silage, corn silage, barley, soybean meal, dried beet pulp, urea, and minerals. In the sugar-rich (SUG) and ethanol-rich (ETH) treatment diets, 20% of DM from CON was replaced with either beets or ensiled beets, respectively. To assess whole-body irreversible loss rate (ILR) and mammary extraction of ethanol and acetate, bilateral jugular catheters were placed for infusion of either [¹³C]-labeled ethanol or acetate, or blood sampling. Additionally, mammary venous blood was sampled by venipuncture. The DMI, milk yield, ethanol content in milk, methane emission, and nitrous oxide content in ruminal headspace gas were unaffected by diet. Plasma flow and mammary net uptake of metabolites, including acetate, ethanol, and amino acids, were also unaffected by diet. Furthermore, feeding 350 g/d of ethanol did not alter the whole-body ILR of ethanol or acetate. The mammary glands were responsible for 57% of the whole-body ILR of ethanol, with a negligible portion secreted into milk. For acetate, the mammary glands were responsible for 39% of the whole-body ILR. In conclusion, feeding 0.35 kg/d of ethanol did not affect the net uptake of ethanol or acetate by the mammary gland or excretion of ethanol in milk. Additionally, feeding either form of beets increased hydrogen emissions but there were no differences in methane emissions among diets.

Summary: The red seaweed Bonnemaisonia hamifera has shown CH₄-mitigating properties in several in vitro studies. This pilot study aimed to investigate the potential of the seaweed as an antimethanogenic feed additive when fed to dairy cows at a maximum inclusion rate of 1% on a dry matter (DM) basis. Increasing the level of B. hamifera resulted in linear decreases in daily CH₄ production, yield, and intensity by 13% to 16%. Moreover, linear increases were observed in daily H₂ emissions, yield, and intensity. Hence, larger and longer-term studies should be undertaken to fully investigate the potential of this seaweed because the concentration of the potentially harmful compound, bromoform, is remarkably lower than in the known antimethanogenic red seaweed Asparagopsis spp.

Limited literature is available identifying phenotypical traits related to enteric methane (CH₄) production from dairy cows, despite its relevance in relation to breeding for animals with a low CH₄ yield (g/kg DMI), and the derived consequences hereof. This study aimed to investigate the relationships between CH₄ yield and different animal phenotypes when 16 2nd parity dairy cows, fitted with a ruminal cannula, were fed 2 diets differing in forage:concentrate ratio in a crossover design. The diets had either a low forage proportion (35% on DM basis, F35) or a high forage proportion (63% on DM basis, F63). Gas exchange was measured by means of indirect calorimetry. Spot samples of feces were collected, and indigestible NDF (INDF) was used as an internal marker to determine total-tract digestibility. In addition, ruminal evacuations, monitoring of chewing activity, determination of ruminal VFA concentration, analysis of relative abundance of methanogens, and measurement of liquid passage rate were performed. Statistical differences were analyzed by a linear mixed model with diet, days in milk, and period as fixed effects, and cow as random effect. The random cow estimates (RCE) were extracted from the model to get the Pearson correlations (r) between RCE of CH₄ yield with RCE of all other variables measured, to identify possible phenotypes related to CH₄ yield. Significant correlations were observed between RCE of CH₄ yield and RCE of OM digestibility (r = 0.63) and ruminal concentration of valeric acid (r = -0.61), acetic acid (r = 0.54), ammonium (r = 0.55), and lactic acid (r = ‒0.53). Additionally, tendencies were observed for correlations between RCE of CH₄ yield and RCE of H₂ yield in g/kg DM (r = 0.47, P = 0.07), and ruminal isobutyric acid concentration (r = 0.43, P = 0.09). No correlations were observed between RCE of CH₄ yield and RCE of ruminal pool sizes, milk data, urinary measurements, or chewing activity. Cows had a lower DMI and ECM, when they were fed F63 compared with F35. Cows fed F63 had higher NDF digestibility, CH₄ emissions (g/d, g/kg of DMI, and g/kg of ECM), ruminal concentration of acetic acid, ruminal pH, degradation rate of digestible NDF (DNDF, %/h), and longer rumen retention time (h). Also, rumination and total chewing time (min/kg DMI) were higher for cows fed F63. The results in the present study emphasize the positive relation between cow's ability to digest OM and their CH₄ emissions. The derived consequences of breeding for lower CH₄ emission might be cows with lower ability to digest OM, but more studies are warranted for further documentation of this relationship.

The 2 brown seaweeds, Fucus serratus and Fucus vesiculosus, have demonstrated anti-methanogenic properties in vitro with reductions in CH₄ production ranging from 53 to 63%. This study aimed to investigate the effects of the 2 Fucus seaweeds on enteric CH₄ emissions, DMI, ECM, and nutrient digestibility when fed to dairy cows. The experiment was conducted using 4 multi-cannulated lactating Danish Holstein dairy cows, which over 3 experimental periods received either: 1) basal diet (CON; diet without any seaweed), 2) basal diet diluted with 4% (DM basis) Fucus serratus (SER), or 3) basal diet diluted with 4% (DM basis) Fucus vesiculosus (VES); resulting in one complete 3 × 3 Latin square and one incomplete 3 × 3 Latin square. Each period lasted 21 d and consisted of 14 d of adaptation, followed by 3 d of digesta sampling, and 4 d of gas exchange measurements using respiration chambers. Milk yield and feed intake were recorded daily. Blood samples were collected on d 15 and 17. All parameters were statistically analyzed using a mixed procedure of R. Opposite to what we had expected, neither of the 2 Fucus seaweeds reduced CH₄ emissions from the dairy cows as daily CH₄ production was significantly higher for both Fucus treatments compared with CON. Additionally, CH₄ yield (g CH₄/kg DMI) and intensity (g CH₄/kg ECM) were significantly higher for SER compared with CON. Milk yield, DMI, and total-tract digestibility were unaffected by the treatments; however, SER resulted in lower milk protein yield (kg/d) and lower milk and blood plasma urea concentrations compared with CON. In conclusion, neither Fucus serratus (SER) nor Fucus vesiculosus (VES) showed potential as methane-mitigating feed additives when fed to dairy cows at an inclusion level of 4% of DM. The inclusion of the 2 brown seaweeds had no effects on DMI, milk yield, or total-tract digestibility.

Summary: Nitrate has been extensively studied and is confirmed to have a significant methane mitigation effect in ruminants. However, it is proven that nitrate supplementation can stimulate the emission of nitrous oxide. In this study, we investigated the effect of nitrate supplementation on ruminal fermentation and related emissions of CH₄, CO₂, and H₂ on cow level, CH₄, CO₂, and N₂O on rumen level, and prediction of N₂O on cow level from rumen volume proportion. The results suggested that nitrate supplementation reduced CH₄ production and yield, with the largest effect on production just after feeding, and increased H₂ production and yield compared with urea supplementation. However, nitrate supplementation also increased N₂O production compared with urea supplementation.

Automated measurements of the ratio of concentrations of methane and carbon dioxide, [CH₄]:[CO₂], in breath from individual animals (the so-called "sniffer technique") and estimated CO₂ production can be used to estimate CH₄ production, provided that CO₂ production can be reliably calculated. This would allow CH₄ production from individual cows to be estimated in large cohorts of cows, whereby ranking of cows according to their CH₄ production might become possible and their values could be used for breeding of low CH₄-emitting animals. Estimates of CO₂ production are typically based on predictions of heat production, which can be calculated from body weight (BW), energy-corrected milk yield, and days of pregnancy. The objectives of the present study were to develop predictions of CO₂ production directly from milk production, dietary, and animal variables, and furthermore to develop different models to be used for different scenarios, depending on available data. An international dataset with 2,244 records from individual lactating cows including CO₂ production and associated traits, as dry matter intake (DMI), diet composition, BW, milk production and composition, days in milk, and days pregnant, was compiled to constitute the training dataset. Research location and experiment nested within research location were included as random intercepts. The method of CO₂ production measurement (respiration chamber [RC] or GreenFeed [GF]) was confounded with research location, and therefore excluded from the model. In total, 3 models were developed based on the current training dataset: model 1 ("best model"), where all significant traits were included; model 2 ("on-farm model"), where DMI was excluded; and model 3 ("reduced on-farm model"), where both DMI and BW were excluded. Evaluation on test dat sets with either RC data (n = 103), GF data without additives (n = 478), or GF data only including observations where nitrate, 3-nitrooxypropanol (3-NOP), or a combination of nitrate and 3-NOP were fed to the cows (GF+: n = 295), showed good precision of the 3 models, illustrated by low slope bias both in absolute values (-0.22 to 0.097) and in percentage (0.049 to 4.89) of mean square error (MSE). However, the mean bias (MB) indicated systematic overprediction and underprediction of CO₂ production when the models were evaluated on the GF and the RC test datasets, respectively. To address this bias, the 3 models were evaluated on a modified test dataset, where the CO₂ production (g/d) was adjusted by subtracting (where measurements were obtained by RC) or adding absolute MB (where measurements were obtained by GF) from evaluation of the specific model on RC, GF, and GF+ test datasets. With this modification, the absolute values of MB and MB as percentage of MSE became negligible. In conclusion, the 3 models were precise in predicting CO₂ production from lactating dairy cows.

Dietary methane (CH₄) mitigation is in some cases associated with an increased hydrogen (H₂) emission. The objective of the present study was to investigate the acute and short-term effects of acceptors for H₂ (fumaric acid, acrylic acid or phloroglucinol) supplemented via pulse-dosing to dairy cows fed CH₄ mitigating diets (using nitrate or 3-nitrooxypropanol), on gas exchange, rumen gas and VFA composition. For this purpose, 2 individual 4 × 4 Latin square experiments were conducted with 4 periods of 3 d (nitrate supplementation) and 7 d (3-nitrooxypropanol supplementation), respectively. In each study, 4 rumen cannulated Danish Holstein cows were used. Each additive for CH₄ mitigation was included in the ad libitum fed diet within the 2 experiments, to which the cows were adapted for at least 14 d. Acceptors for H₂ were administered twice daily in equal portions through the rumen fistula immediately after feeding of the individual cow. In Exp. 1 (nitrate), the treatments were CON-1 (no H₂-acceptor), FUM-1 (fumaric acid), ACR-1 (acrylic acid) and FUM+ACR-1 (50% FUM-1 + 50% ACR-1). In Exp. 2 (3-nitrooxypropanol), the 3 treatments, CON-2, FUM-2, and ACR-2, were similar to CON-1, FUM-1 and ACR-1 treatments, however the fourth treatment was PHL-2 (phloroglucinol). Gas exchanges were measured in respiration chambers, while samples of rumen liquid and headspace gas were taken in time series relative to feeding and dosing on specific days. Headspace gas was analyzed for gas composition and rumen liquid was analyzed for volatile fatty acid composition and dissolved gas concentrations. Headspace gas composition and dissolved gas concentration were only measured in Exp. 2. Dry matter intake was reduced upon acrylic acid supplementation. There were no significant effects of any treatments in any experiments on H₂ emission, except for a decrease in hourly H₂ emission rate (g/h) at 1 h after feeding in both experiments. In Exp. 2, H₂ headspace proportions increased by ACR-2 supplementation, whereas dissolved concentrations were unaffected. In Exp. 1, cows on ACR-1 increased propionate proportion at 1 h after feeding. In Exp. 2, both FUM-2 and ACR-2 increased rumen propionate proportion in the hours after feeding and dosing. There was no effect on rumen acetate for cows on PHL-2. There was a strong positive correlation between rumen dissolved CH₄ and headspace CH₄ (r = 0.84), whereas the equivalent correlation was weaker for H₂ (r = 0.41). For the relationship between dissolved concentrations and emissions of CH₄ and of H₂, there was a moderate positive correlation for CH₄ (r = 0.54), whereas it was weak for H₂ (r = 0.28) with zero slope. In conclusion, the results suggested that fumaric acid and acrylic acid to some extent was reduced to propionate without associative effects on measures for H₂ redirection. Furthermore, phloroglucinol seemed not to be metabolized in the rumen in the present study, as no effects on rumen acetate or measures of H₂ were observed. Changes in H₂ headspace and emission may be a poor proxy for actual changes in the rumen fluid concentration of H₂.

Fat in the form of cracked rapeseed and 3-nitrooxypropanol (3-NOP, market as Bovaer®) were fed alone or in combination to 4 Danish Holstein multi-cannulated dairy cows, with the objective to investigate effects on gas exchange, dry matter intake (DMI), nutrient digestion, and nutrient metabolism. The study design was a 4 × 4 Latin square with a 2 × 2 factorial treatment arrangement with 2 levels of fat supplementation; 33 g crude fat/kg of dry matter (DM) or 64 g crude fat/kg of DM for low and high fat diets, respectively, and 2 levels of 3-NOP; 0 mg/kg of DM or 80 mg/kg of DM. In total, 4 diets were formulated: low fat (LF), high fat (HF), 3-NOP and low fat (3LF), and 3-NOP and high fat (3HF). Cows were fed ad libitum and milked twice daily. The adaptation period lasted 11 d, followed by 5 d with 12 diurnal sampling times of digesta and ruminal fluid. Thereafter, gas exchange was measured for 5 d in respiration chambers. Chromic oxide and titanium dioxide were used as external flow markers to determine intestinal nutrient flow. No interactions between fat supplementation and 3-NOP were observed for methane yield (g/kg of DM), total-tract digestibility of nutrients or total volatile fatty acid (VFA) concentration in the rumen. Methane yield (g/kg of DMI) was decreased by 24% when cows were fed 3-NOP. In addition, 3-NOP increased carbon dioxide and hydrogen yield (g/kg of DM) by 6% and 3,500%, respectively; However, carbon dioxide production was decreased when expressed on a daily basis. Fat supplementation did not affect methane yield but tended to reduce methane in percent of gross energy intake. A decrease (11%) in DMI was observed, when cows were fed 3-NOP. Likely, the lower DMI mediated a lower passage rate causing the tendency to higher rumen and total-tract neutral detergent fiber digestibility, when the cows were fed 3-NOP. Total VFA concentrations in the rumen were negatively affected both by 3-NOP and fat supplementation. Furthermore, 3-NOP caused a shift in the VFA fermentation profile, with decreased acetate proportion and increased butyrate proportion, whereas propionate proportion was unaffected. Increased concentrations of the alcohols methanol, ethanol, propanol, butanol, and 2-butanol were observed in the ruminal fluid when cows were fed 3-NOP. These changes in rumen metabolites indicate partial re-direction of hydrogen into other hydrogen sinks, when methanogenesis is inhibited by 3-NOP. In conclusion, fat supplementation did not reduce methane yield, whereas 3-NOP reduced methane yield, irrespective of fat level. However, the concentration of 3-NOP and diet composition and resulting desired mitigation effect must be considered before implementation. The observed reduction in DMI with 80 mg 3-NOP/kg DM was intriguing and may indicate that a lower dose should be applied in a Northern European context; however, the mechanism behind needs further investigation.