Hanne Helene Hansen

Iodoform has been shown to be an enteric methane-reducing agent. This study aimed to investigate if iodoform differentially affected maize (MS) and clover grass silage (GS). These substrates were used in four 22 h in vitro gas production fermentations with and without 0.032 mg/g of dry matter substrate of 0.064% iodoform in zeolite (IZ). The results of organic matter degradation (dOM, g/g), volatile fatty acids (VFAs, mMol/L), partitioning factor (PF, mg/mL), total gas production (TGP, mL/g OM), and periodic hydrogen and methane yield (mL/g OM) were evaluated. The interaction between substrate and IZ for dOM was significant with significantly less in GS. The interaction between substrate and additive was continuously significant for TGP. The additive in GS numerically decreased the TGP, but significantly increased TGP in MS. The interaction between substrate and additive was sporadically significant with methane, increasing the yield when IZ was added to MS, but a consistent, non-significant, decrease in yield was found when IZ was added to GS. Total VFA production and propionate content increased in both substrates with the addition of IZ, but only significantly in GS. Iodoform significantly decreased the efficiency of microbial conversion (PF) in both substates. Differential effects of iodoform on GS and MS were evident. More research is needed to determine the modes of action of iodoform.

Background: This study aimed to investigate the impact of in vitro rumen fermentation (IVRF) on the microbiome structure and composition of rumen fluid before and after fermentation assays. Methods and Results: Six separate fermentation batches were run for 48 h using maize silage as the basal feed. Rumen fluid samples were analyzed before (RF; only rumen fluid inoculant) and after 48 h fermentation assay (MS; maize silage as the substrate) and further processed for microbiome analysis using amplicon sequencing targeting the V4 region of the bacterial 16S rRNA gene. Bacterial alpha diversity revealed that the Shannon index and observed index were similar between MS and RF fluid. The core microbiome was detected in 88.6% of the amplicon sequence variants in MS and RF. Taxonomic analysis at the phylum level showed similar abundances of Bacteroidetes, Proteobacteria, Firmicutes, Verrucomicrobiota, Spirochaetota, Patescibacteria, and Campilobacterota in MS and RF. The Bray–Curtis distance matrix showed similar bacterial community structure among MS and RF samples. Conclusion: Our results indicated that the in vitro procedure did not affect the bacterial community structure compared to the original rumen fluid inoculum. It should be noted that assessing the microbiome at a single endpoint (i.e., 48 h) may not provide a comprehensive understanding of the microbiome profile dynamics. However, the findings of this study provide a basis for future microbiome-based in vitro fermentation tests and confirm that the technique allows a high degree of species diversity that approximates the rumen function in vivo.

In vitro gas production techniques (IVGPT) are widely used to screen feeds and feed additives to reduce the number of animals needed for experiments, which in turn, reduces costs and increases animal welfare. However, information about repeatability is scarce. The objective of this study was to evaluate the variation from in vitro gas production fermentations in the same laboratory using the same feed substrate. The source of rumen fluid used in the fermentations was from two different farms with either cannulated lactating dairy cows or cannulated fasting heifers, representing two distinct stages of production (donor types). Seventeen 24 h fermentations, undertaken during a year, were used to evaluate the variation between the following parameters: gas curve parameters, baseline-corrected total gas production (TGP (mL at Standard Temperature and Pressure (STP))/g incubated dry matter (DM)), methane concentration (%) and yield (mL gas at STP/g DM), pH and degraded dry matter (dDM). Significant differences between donor types were found for the pH of the rumen fluid from individual animals and pH of fermented fluid. However, no significant differences were observed within donor type. The means for methane concentration and yield, after 24 h of fermentation, were not significantly different between or within donor types. Rate of early gas production was significantly different between donor types, but baseline-corrected TGP was not significantly different at 24 h. No dDM differences after 24 h of fermentation between or within donor types were detected. Gas production curves were different between donor types, being either a monophasic version of the sigmoidal model or an exponential curve for the heifers and the production animals, respectively. No differences were observed within type. Repeatability of rumen fluid (CV_RF), calculated as the coefficient of variation, and the associated parameters, which were investigated, was best for methane yield (CV_RFALL = 0.3%) and least for TGP at 3 h (CV_RFALL = 3%). Repeatability was dependent on donor type.

Direct-fed microbial products (DFM) are probiotics that can be used advantageously in ruminant production. The in vitro gas production technique (IVGPT) is a method to simulate rumen fermentation and can be used to measure degradation, gas production, and products of fermentation of such additives. However, inter-laboratory differences have been reported. Therefore, tests using the same material were used to validate laboratory reproducibility. The objective of this study was to assess the effect of adding two DFM formulations on fermentation kinetics, methane (CH₄) production, and feed degradation in two different basal feeds while validating a newly established IVGPT laboratory. Six treatments, with three replicates each, were tested simultaneously at the established IVGPT lab at the University of Copenhagen, and the new IVGPT lab at Chr. Hansen Laboratories. Maize silage (MS) and grass silage (GS) were fermented with and without the following DFM: P1: Ligilactobacillus animalis and Propionibacterium freudenreichii (total 1.5 × 10⁷ CFU/mL), P2: P1 with added Bacillus subtilis and B. licheniformis (total 5.9 × 10⁷ CFU/mL). The DFM were anaerobically incubated in rumen fluid and buffer with freeze-dried silage samples for 48 h. Total gas production (TGP: mL at Standard Temperature and Pressure/gram of organic matter), pH, organic matter degradability (dOM), CH₄concentration (MC) and yield (MY), and volatile fatty acid (VFA) production and profiles were measured after fermentation. No significant differences between the laboratories were detected for any response variables. The dOM of MS (78.3%) was significantly less than GS (81.4%), regardless of the DFM added (P1 and P2). There were no significant differences between the effects of the DFM within the feed type. MS produced significantly more gas than GS after 48 h, but GS with DFM produced significantly more gas at 3 and 9 h and a similar gas volume at 12 h. Both DFM increased TGP significantly in GS at 48 h. There was no difference in total VFA production. However, GS with and without probiotics produced significantly more propionic acid and less butyric acid than MS with and without probiotics. Adding P2 numerically reduced the total methane yield by 4–6% in both MS and GS. The fermentation duration of 48 h, used to determine maximum potential dOM, may give misleading results. This study showed that it is possible to standardize the methodology to achieve reproducibility of IVGPT results. Furthermore, the results suggest that the P2 DFM may have the potential to reduce CH₄ production without affecting organic matter degradation.

The objective of this study was to determine the effect of autochthonous Nepalese fruits on nutrient degradation, fermentation kinetics, total gas production, and methane production in in-vitro rumen fermentation. The fruits of Terminalia chebula (HA), Terminalia bellirica (BA), and Triphala churna (TC), a commercial mixture with equal parts (33.3% DM basis) of Phyllanthus emblica, Terminalia bellirica, and Terminalia chebula, were used. These were tested at three inclusion levels of 20% 40% and 100% of the total sample (as dry matter) in maize silage (MS). MS was used as a control (0% additive). These 10 treatments were tested for two 48-h incubations with quadruplicate samples using rumen fluid from 2 heifers. Total gas production (TGP: mL at standard temperature and pressure (STP)/g DM), methane production (expressed as % and mL/g DM), and volatile fatty acids were determined. After incubations, the filtrate was used to measure pH and volatile fatty acids (VFA), while the residue was used to measure degraded dry matter (dDM) and calculate the partitioning factor (PF48) and theoretical short-chain fatty acid concentration (tVFA). Rumen fluid pH linearly (p < 0.01) decreased in all treatments with increasing dose during fermentation. The CH₄% was less in all three treatments with 100% autochthonous plants than in control, but there were no significant linear or quadratic effects for increasing BA, HA, and TC doses. The PF48 increased for all treatments with a significant linear and quadratic effect (p < 0.05) of increasing dose. Compared to MS, the inclusion of autochthonous plants increased the total volatile fatty acids, with no significant dose effects. The tVFA linearly decreased (p > 0.05) with an increasing dose of BA and HA. All treatments showed quadratic effects on tVFA (p < 0.05) with increasing dose. Increasing TC dose linearly (p < 0.05) and quadratically (p < 0.05) increased total VFA, while increasing HA dose had only a quadratic (p < 0.05) effect on total VFA. All treatments reduced total gas production (TGP) and methane concentration (CH₄%) when compared to MS. The tested autochthonous fruits can be used as additives with a basal feed diet to reduce enteric methane emissions. The most effective anti-methanogenic treatment was 40% HA, which resulted in 18% methane reduction.

The aim of this study was to determine the effect of supplementing dairy goat diets with rapeseed oil and sunflower oil on performance, milk composition, milk fatty acid profile, and in vitro fermentation kinetics. Nine Danish Landrace goats with 42 ± 5 days in milk were allocated to three treatment groups for 42 days. Animals received a basal diet, formulated with 85:15 forage:concentrate ratio, and the basal diet was supplemented with either rapeseed oil or sunflower oil at 4% of dry matter. Goat milk was sampled on days 14, 21, and 42. Milk composition was similar between treatments. From day 14 to day 42, milk yield increased (1.03 vs. 1.34 kg/d), while milk fat (2.72 vs. 1.82 g/d) and total solids (11.2 vs. 9.14 %) were reduced. Compared to control and rapeseed oil, sunflower decreased (P < 0.05) C4:0 (1.56, and 1.67 vs. 1.36 g/100 g) and both oils decreased (P < 0.05) C18:3n3 (0.60 vs. 0.20 and 0.10 g/100g). Rapeseed oil increased (P < 0.05) C18:2 cis9, trans11 compared to control and sunflower oil (0.37 vs. 0.13 and 0.19 g/100 g). Untargeted milk foodomics revealed slightly elevated (P < 0.05) gluconic acid and decreased hippuric acid (P < 0.05) in the milk of oil-fed goats compared to control. In vitro dry matter degradation (63.2 ± 0.02 %) was not affected by dietary treatments, while individual volatile fatty acid proportions, total volatile fatty acids (35.7 ± 2.44 mmol/l), CO₂ (18.6 ± 1.15 mol), and CH₄ (11.6 ± 1.16 mol) were not affected by dietary treatments. Sunflower oil and rapeseed oil decreased (P < 0.05) total gas production at 24 and 48 h compared with control. Overall, the use of sunflower oil or rapeseed oil at 4% DM inclusion did not compromise animal performance and milk composition.

The dietary supplementation of vegetable oils is known to improve the dietary energy density as well as milk fatty acid profile; however, the impacts on the milk foodome is largely unknown. This study investigated the effect of two different sources of unsaturated fatty acids, rapeseed oil and sunflower oil, as a feeding supplement on the milk foodome from dairy goats. Nine Danish Landrace goats at 42 ± 5 days in milk were allocated to three treatment groups for 42 days with three animals per group. A control group received a basal diet made of forage and concentrate at an 85:15 ratio. On top of the basal diet, the second and third groups received rapeseed oil or sunflower oil supplements at 4% of dry matter, respectively. Goat milk was sampled on days 14, 21, and 42. The milk foodome was measured using gas chromatography–mass spectrometry and proton nuclear magnetic resonance spectroscopy. The milk levels of 2-hydroxyisovaleric acid, oxaloacetic acid, and taurine were higher in the milk from goats fed with sunflower oil compared to the control group. More glucose-1-phosphate was found in the milk from goats fed with rapeseed oil compared to the control group. Amino acids, valine and tyrosine, and 2-hydroxyisovaleric acid and oxaloacetic acid were higher in the sunflower group compared to the rapeseed group, while the milk from the rapeseed-fed goats had greater levels of ethanol and 2-oxoglutaric acid compared to the sunflower group. Thus, results show that foodomics is suitable for studying how milk chemistry changes as a function of feeding regime.

Replacement of conventional feedstuffs with inexpensive and non-conventional ingredients such as quinoa may improve animal performance and the quality of their products. Quinoa supplementation is believed to have a good nutritive value as a ruminant feed, but evidence is scarce. The present experiment aimed to evaluate the nutritive value of whole, dried quinoa plant (Chenopodium quinoa) as a feed for ruminants. In the first experiment, the in sacco technique was used to evaluate nutrient disappearance and fermentation kinetics of quinoa. In the second experiment, the in vitro gas production technique was used to evaluate diets with substitution of clover hay with quinoa at 0 (Q0), 15 (Q15), 30 (Q30), and 45% (Q45) of the diets. Proximate analysis showed that quinoa contained about 18.6% crude protein (CP) with oleic acid, arachic acid, linoleic acid, and palmitic acid as the major fatty acids. The in sacco degradability showed that the “a” fraction of dry matter (DM) was low, while the fraction “b” was high for DM and CP. Replacing clover hay with quinoa did not affect gas or methane production; however, Q30 treatment quadratically increased (P < 0.05) its production. It is concluded that quinoa can be used as a feed for ruminants and can replace clover hay up to 45% in the diet.