Evaluation of ruminal methane and ammonia formation and microbiota composition as affected by supplements based on mixtures of tannins and essential oils using Rusitec.

BACKGROUND: Dietary supplements based on tannin extracts or essential oil compounds (EOC) have been repeatedly reported as a promising feeding strategy to reduce the environmental impact of ruminant husbandry. A previous batch culture screening of various supplements identified selected mixtures with an enhanced potential to mitigate ruminal methane and ammonia formation. Among these, Q-2 (named after quebracho extract and EOC blend 2, composed of carvacrol, thymol, and eugenol) and C-10 (chestnut extract and EOC blend 10, consisting of oregano and thyme essential oils and limonene) have been investigated in detail in the present study with the semi-continuous rumen simulation technique (Rusitec) in three independent runs. For this purpose, Q-2 and C-10, dosed according to the previous study, were compared with a non-supplemented diet (negative control, NC) and with one supplemented with the commercial EOC-based Agolin® Ruminant (positive control, PC). RESULTS: From d 5 to 10 of fermentation incubation liquid was collected and analysed for pH, ammonia, protozoa count, and gas composition. Feed residues were collected for the determination of ruminal degradability. On d 10, samples of incubation liquid were also characterised for bacterial, archaeal and fungal communities by high-throughput sequencing of 16S rRNA and 26S ribosomal large subunit gene amplicons. Regardless of the duration of the fermentation period, Q-2 and C-10 were similarly efficient as PC in mitigating either ammonia (-37% by Q-2, -34% by PC) or methane formation (-12% by C-10, -12% by PC). The PC was also responsible for lower feed degradability and bacterial and fungal richness, whereas Q-2 and C-10 effects, particularly on microbiome diversities, were limited compared to NC. CONCLUSIONS: All additives showed the potential to mitigate methane or ammonia formation, or both, in vitro over a period of 10 d. However, several differences occurred between PC and Q-2/C-10, indicating different mechanisms of action. The pronounced defaunation caused by PC and its suggested consequences apparently determined at least part of the mitigant effects. Although the depressive effect on NDF degradability caused by Q-2 and C-10 might partially explain their mitigation properties, their mechanisms of action remain mostly to be elucidated.

Comparative assessment of emulsifiers for in vitro ruminal gas production and fermentation measurements: Tween 80 is a suitable emulsifier.

Emulsifiers are essential for achieving a homogenous distribution of lipophilic supplements in in vitro rumen fluid incubations. Since emulsifiers can alter rumen fermentation, it is crucial to select one that minimally impacts fermentation parameters to reduce potential biases. This study aimed to evaluate seven emulsifiers' impact on in vitro ruminal fermentation using the Hohenheim Gas Test in order to identify the most inert emulsifier. Rumen fluids were collected from three non-lactating Original Brown-Swiss cannulated cows before morning feeding and incubated for 24 h with a basal diet in triplicates. The emulsifiers tested were ethanol, ethyl acetate, propylene glycol, glycerol, ethylene glycol, soy lecithin, and Tween® 80, each in two dosages (0.5% or 1% v/v). The untreated basal diet served as control. Compared to control, in vitro organic matter digestibility was enhanced by ethyl acetate (by 36.9 and 48.2%), ethylene glycol (by 20.6 and 20.1%), glycerol (by 46.9 and 56.8%) and soy lecithin (by 19.7 and 26.8%) at 0.5 and 1% dosage, respectively. Additionally, the 24-h methane production increased for ethanol (by 41.9 and 46.2%), ethylene glycol (by 50.5 and 51.5%), and glycerol (by 63.1 and 65.4%) for the 0.5 and 1% dosage, respectively, and 0.5% dosage for ethyl acetate (by 31.6%). The acetate molar proportion was 17.2%pt higher for ethyl acetate, and 25.5%pt lower for glycerol at 1% dosage, compared to the control. The propionate concentration was 22.1%pt higher 1% glycerol, and 15.2%pt and 15.1%pt higher for 0.5 and 1% propylene glycol, respectively, compared to the control. In summary, Tween® 80 did not significantly affect in vitro rumen fermentation parameters, making it the most suitable choice for in vitro incubations involving lipophilic substances in rumen fluid. Ethanol may be considered as an alternative emulsifier if methane production is not the variable of interest.

Evaluation and quantification of associations between commonly suggested milk biomarkers and the proportion of grassland-based feeds in the diets of dairy cows.

This study is a first step approach towards the prediction of the proportion of grassland-based feeds (%GB) in dairy cow diets with the aid of three different groups of milk biomarkers. We aimed to evaluate and quantify the associations between biomarkers commonly suggested in the literature and %GB in individual cows as a hypothesis-generating stage for the prospective establishment of accurate %GB prediction models. Consumers and governments financially encourage sustainable, local milk production making grass-based feeding, in grassland-dominated regions, of major interest. Milk from grassland-fed cows differs from that of other feeding systems by inferential fatty acids (FA), ß-carotene content and yellow color; however, these biomarkers have not been evaluated together for their association with %GB. Using approved methods of parametric regression analysis, gas chromatography (GC), mid-infrared spectra (MIR) and color spectroscopy, we aimed to develop a first step towards an easy-to-implement, cost-effective milk-based control to estimate %GB in dairy cow diets. The underlying database was generated with 24 cows each fed one of 24 different diets gradually increasing in grass silage and decreasing in corn silage. Our results indicate that GC-measured α-linolenic acid, total n-3 FA and the n-6:n-3 ratio, MIR-estimated PUFA and milk red-green color index a* are robust milk biomarkers for constructing accurate prediction models to determine %GB. Based on simplified regression analysis, diets containing 75% GB should contain ≥ 0.669 and 0.852 g α-linolenic acid and total n-3 FA per 100 g total FA, respectively, and an n-6:n-3 FA ratio of < 2.02 measured with GC; estimated with MIR, polyunsaturated FA should be ≥ 3.13 g/100 g total FA. ß-carotene was not a good predictor for estimating %GB. Unexpectedly, the milk became greener with increasing %GB (negative a* values, ‒6.416 for 75% GB), suggesting the red-green color index, not yellow-blue, as a suitable biomarker.

A pilot investigation on the effect of induced saliva flow on digestive parameters in sheep, and a comparison with cattle.

Sheep with a relatively low methane yield were observed to have shorter fluid and particle mean retention times (MRT). Because the application of pilocarpine, a saliva stimulant, was successful in reducing retention times in ruminants in previous studies, we applied this substance to sheep, expecting a reduction in MRT and methane yield. Three non-pregnant sheep (74 ± 10 kg) were fed a hay-only diet in a 3 × 3 Latin square design with oral doses of 0, 2.5 and 5 mg pilocarpine/kg body weight and day. Measurements included feed and water intake, MRT of liquid and particulate phases in the reticulorumen (RR) and total gastrointestinal tract (GIT), ruminal microbial yield (via urinary purine bases and metabolic faecal nitrogen), total tract methane emission, apparent nutrient digestibility and rumen fluid parameters. Data were investigated for linear and quadratic effects using orthogonal polynomial contrasts. The MRT of liquid and small particles in the RR and total GIT, and the short-chain fatty acid concentration in rumen fluid, linearly declined with increasing pilocarpine dosage, while no quadratic relationship was detected. Intake of feed DM and water, apparent nutrient digestibility, methane yield and microbial yield were not affected by pilocarpine. When combining the sheep data with that of a similar experiment in cattle, we found that the MRT of the liquid phase was positively associated with estimated NDF digestibility and with methane production per digested NDF, but was not associated with microbial yield or the ratio of acetate to propionate. The ratio between MRT of the particulate and the liquid phase was smaller for sheep than that for cattle, and was not affected by treatment. Differences in this ratio might explain why species reacted differently to the saliva-inducing agent, which might help to explain the discrepancy between species in the effect of induced saliva flow on digestive parameters.

Effects of incremental increases in grass silage proportions from different harvest years on methane emissions, urinary nitrogen losses, and protein and energy utilisation in dairy cows.

Dairy cows, methane and global warming have become publicly related terms. However, appropriate dairy cow management may in fact be part of the climate solution when viewed as part of the biogenic carbon cycle. Accordingly, governments and consumers are encouraging more sustainable, locally produced, climate friendly dairy production that often includes grassland-based feeding. However, this system is presumed to result in greater methane emissions compared to corn silage- or concentrate-based diets. An increase in urine nitrogen, associated with increased ammonia and nitrous oxide emission potential, questions the environmental usefulness of this strategy pertaining to global warming. This study is the first to compare the effects of incremental increases of grass silage proportion on enteric methane production as well as N and energy losses in dairy cows. Twenty-four mid- to end-lactation dairy cows were each fed one of 24 different diets, from two different harvest years, gradually increasing in grassland-based feeds (grass silage and hay) from about 500 to 1000 g/kg and concomitantly decreasing in corn silage. Each cow underwent a 7-day total collection period and was housed for 48 h in respiration chambers. Incremental data were subjected to an approved parametric regression analysis approach. The dietary increase in grassland-based feeds did not impair milk yield, N and energy utilisation. Simplified regression equations revealed that, contrary to current assumptions, there was a decline in methane production from 373 to 303 g/day when increasing grassland-based feeds from 500 to 1000 g/kg diet, and there was a trend for a decline in emission intensity from 20.6 to 17.6 g/kg of energy-corrected milk. However, urine nitrogen emissions clearly increased even when related to nitrogen intake; the latter from 260 to 364 g/kg when increasing grassland-based feeds from 500 to 1000 g/kg. Methane and urine nitrogen emissions were not affected by year of harvest.

Individual differences in digesta retention and their relation to chewing in cattle-A pilot investigation.

While information on individual differences in digesta mean retention time (MRT) might be interesting when selecting phenotypes for digestive efficiency, MRT measurements are prohibitively labour-intensive for large-scale application. Therefore, more easily measured proxies of MRT might be helpful. We used the opportunity of an experiment applying saliva stimulant in cattle to investigate the effect of different individual chewing behaviour on fluid and particle MRT with a consistent diet. Four non-lactating cattle (670-850 kg body mass [BM]) were used in a 4 × 4 Latin square design, treated with the saliva stimulant pilocarpine in dosages of 0, 1, 2.5 and 5 mg/kg BM per day. The cattle were fed hay with dry matter intake (DMI) assigned according to their metabolic body weight. MRT in the whole gastrointestinal tract (GIT), the reticulorumen (RR) and the distal tract were measured using Co-EDTA, Cr-mordanted fibre and La-mordanted fibre as markers representing fluid, small particles (2 mm) and large particles (1 cm), respectively. The chewing behaviour was measured via noseband pressure sensor and expressed as chewing frequency (chews per time) and chewing intensity (chews per DMI), both for total chewing (ingestion plus rumination) and rumination chewing alone. The animals differed considerably in chewing behaviour and MRT measures. BM did not show a significant effect on chewing behaviour and MRT measures, though it tended to negatively correlated to total chewing intensity. Chewing intensity exerted a significant negative influence on MRT of fluid and particles in the RR, which was not the case for chewing frequency. Chewing frequency showed a significant relationship with MRT of large particles in the GIT. We suggest that chewing behaviour could influence MRT in two ways: (i) by affecting saliva production via the masticatory-salivary reflex and subsequently, the fluid inflow to the RR; (ii) by contributing to particle size reduction. Should the link between chewing behaviour and MRT be corroborated in larger studies, chewing measures, with their large interindividual variation, could emerge as an easy-to-measure proxy for MRT characteristics.

Effect of induced saliva flow on fluid retention time, ruminal microbial yield and methane emission in cattle.

Both in vitro and animal studies indicated that a higher dilution rate is related to a more efficient microbial synthesis and a lower methane (CH4 ) yield. The latter could be a consequence of the former, as an increase in microbial cell synthesis offers an alternative hydrogen sink competing with methanogenesis. To test this assumption in live animals, we applied a saliva stimulant, pilocarpine, to modify liquid flow rate in cattle. Four non-lactating cows (750 ± 71 kg) were fed forage only (restricted to constant intake) in a 4 × 4 Latin square design with oral doses of 0, 1, 2.5 and 5mg pilocarpine/kg body weight and day. We quantified feed and water intake, ruminal and total tract mean retention time (MRT) of solute and particle markers, ruminal microbial yield (via urinary purine bases or metabolic faecal nitrogen), CH4 emission, digestibility, chewing behaviour, reticular motility and rumen fluid parameters. The effect of induced saliva flow was evident by visibly increased salivation and water intake. Increasing the pilocarpine dosages resulted in a linearly decreased MRT of fluid and small particles (p < 0.001 and p< 0.05, respectively) and methane yield as related to digested DM (p < 0.05), the latter at a magnitude of 5%. No effect of treatment was found on ruminal microbial yield estimated via purine derivates. Metabolic faecal N as an indicator of microbial growth linearly correlated with pilocarpine dosages (p < 0.05). No significant relationship was found between pilocarpine dosages and large particle MRT, nutrient digestibility, ruminal pH and short-chain fatty acids. In conclusion, different from some in vitro studies, there was little indication of a reciprocal effect of CH4 and microbial biomass production in cows fed a forage-only diet.

Genotype-by-Diet Interactions for Larval Performance and Body Composition Traits in the Black Soldier Fly, Hermetia illucens.

Further advancing black soldier fly (BSF) farming for waste valorisation and more sustainable global protein supplies critically depends on targeted exploitation of genotype-phenotype associations in this insect, comparable to conventional livestock. This study used a fully crossed factorial design of rearing larvae of four genetically distinct BSF strains (FST: 0.11-0.35) on three nutritionally different diets (poultry feed, food waste, poultry manure) to investigate genotype-by-environment interactions. Phenotypic responses included larval growth dynamics over time, weight at harvest, mortality, biomass production with respective contents of ash, fat, and protein, including amino acid profiles, as well as bioconversion and nitrogen efficiency, reduction of dry matter and relevant fibre fractions, and dry matter loss (emissions). Virtually all larval performance and body composition traits were substantially influenced by diet but also characterised by ample BSF genetic variation and, most importantly, by pronounced interaction effects between the two. Across evaluated phenotypes, variable diet-dependent rankings and the lack of generally superior BSF strains indicate the involvement of trade-offs between traits, as their relationships may even change signs. Conflicting resource allocation in light of overall BSF fitness suggests anticipated breeding programs will require complex and differential selection strategies to account for pinpointed trait maximisation versus multi-purpose resilience.

In vitro ruminal fermentation, methane production and nutrient degradability as affected by fruit and vegetable pomaces in differing concentrations.

Pomaces are food industry by-products and may serve as animal feed to increase sustainability of meat and milk production. The aim of the present study was to evaluate fermentation characteristics of dried fruit and vegetable pomaces in a short-term in vitro experiment using the Hohenheim Gas Test. A selection of six fruit (apple, aronia, orange, pomegranate, red, white grape) and three vegetable (beetroot, carrot, tomato) pomaces was tested in three concentrations (150, 300, 500 g kg-1 of dry matter (DM)) as supplement to the basal diet (hay, used as control). Three runs were performed, using rumen fluid from one of three different rumen-cannulated cows in each run. Per run, each compound was tested in duplicate. After 24 h incubation, total gas production, methane and CO2 concentration, short-chain fatty acids, in vitro organic matter digestibility as well as microbial counts were determined. In addition, the pomaces' polyphenol content including the fractions non-tannin phenols, condensed tannins and hydrolysable tannins were analysed. Most pomaces did not significantly affect rumen fermentation characteristics in any of the tested dosages and may thus be applied in ruminant nutrition without adverse effects. Aronia significantly decreased (-14.5%) the organic matter digestibility in the highest concentration whereas apple (+12%), carrot (+10%) and beetroot (+8%) increased gas formation related to digestible organic matter. The 500 g kg-1 dosage of pomegranate significantly decreased methane formation by about 28% without impairing digestibility. Pomegranate was the only pomace of those high in total tannins that contained exceptionally high amounts of hydrolysable (90% of total tannins) and proportionally low amounts of condensed tannins (10% of total tannins), indicating that the hydrolysable tannins most likely reduced the methane production. Therefore, pomegranate pomace may be an interesting option for a methane mitigating feed supplement in ruminants and should be considered for following in vivo testing.

The rumen microbiome inhibits methane formation through dietary choline supplementation.

Enteric fermentation from ruminants is a primary source of anthropogenic methane emission. This study aims to add another approach for methane mitigation by manipulation of the rumen microbiome. Effects of choline supplementation on methane formation were quantified in vitro using the Rumen Simulation Technique. Supplementing 200 mM of choline chloride or choline bicarbonate reduced methane emissions by 97-100% after 15 days. Associated with the reduction of methane formation, metabolomics analysis revealed high post-treatment concentrations of ethanol, which likely served as a major hydrogen sink. Metagenome sequencing showed that the methanogen community was almost entirely lost, and choline-utilizing bacteria that can produce either lactate, ethanol or formate as hydrogen sinks were enriched. The taxa most strongly associated with methane mitigation were Megasphaera elsdenii and Denitrobacterium detoxificans, both capable of consuming lactate, which is an intermediate product and hydrogen sink. Accordingly, choline metabolism promoted the capability of bacteria to utilize alternative hydrogen sinks leading to a decline of hydrogen as a substrate for methane formation. However, fermentation of fibre and total organic matter could not be fully maintained with choline supplementation, while amino acid deamination and ethanolamine catabolism produced excessive ammonia, which would reduce feed efficiency and adversely affect live animal performance.

Transfer of Lauric and Myristic Acid from Black Soldier Fly Larval Lipids to Egg Yolk Lipids of Hens Is Low.

Implementing insects, such as the black soldier fly larvae (BSFL), as animal feed commonly includes the previous removal of substantial amounts of fat. This fat may represent an as yet underutilized energy source for livestock. However, transfer of lauric and myristic acid, prevalent in BSFL fat and undesired in human nutrition, into animal-source foods like eggs may limit its implementation. To quantify this, a laying hen experiment was performed comprising five different diets (10 hens/diet). These were a control diet with soybean oil and meal and a second diet with soybean oil but with partially defatted BSFL meal as protein source. The other three diets were based on different combinations of partially defatted BSFL meal and fat obtained by two different production methods. Lauric acid made up half of the BSFL fat from both origins. Both BSFL fats also contained substantial amounts of myristic and palmitic acid. However, in the insect-based diets, the net transfer from diet to egg yolk was less than 1% for lauric acid, whereas the net transfer for myristic and palmitic acid was about 30% and 100%, respectively. The net transfer did not vary between BSFL originating from production on different larval feeding substrates. The results illustrate that hens are able to metabolize or elongate very large proportions of ingested lauric acid and myristic acid, which are predominant in the BSFL lipids (together accounting for as much as 37 mol%), such that they collectively account for less than 3.5 mol% of egg yolk fatty acids.

Supplementation of Pelleted Hazel (Corylus avellana) Leaves Decreases Methane and Urinary Nitrogen Emissions by Sheep at Unchanged Forage Intake.

This study is the first to quantify the effects of hazel (Corylus avellana) leaves on methane and urinary nitrogen emissions, digestibility, nitrogen and the energy balance of ruminants. Four experimental pellets were produced with 0, 30% and 60% hazel leaves, the latter also with 4% polyethylene glycol. Hazel leaves gradually replaced lucerne. The diet was composed of the pellets and grass hay (80%: 20%). Six adult sheep were allocated to all four treatments in a 6 × 4 crossover design. Including hazel leaves did not affect the feed intake, but it decreased the apparent digestibility of organic matter and fibre, especially at the high level. Methane emission was reduced by up to 25 to 33% per day, per unit of intake and per unit of organic matter digested. Urinary nitrogen excretion decreased by 33 to 72% with increasing levels of hazel leaves. The treatment with polyethylene glycol demonstrated that tannins in hazel leaves caused significant shares of the effects. In conclusion, the current results indicated a significant potential of hazel leaves as forage for ruminants to mitigate methane and urinary nitrogen emissions. Even high dietary hazel leaf proportions were palatable. The lower digestibility needs to be compensated with easily digestible diet ingredients.